Methods of treating mild brain injury

ABSTRACT

The present disclosure provides methods for treating mild brain injury and other neurological disorders in a subject, comprising administering to the subject an effective amount of a compound comprising ghrelin.

CROSS-REFERENCE

This application is a continuation of application Ser. No. 16/844,934,filed Apr. 9, 2020, which application is a continuation of applicationSer. No. 16/143,735, filed Sep. 27, 2018, which application is acontinuation of application Ser. No. 15/114,067, filed Jul. 25, 2016(now U.S. Pat. No. 10,105,416), which is a U.S. National StageApplication filed under 35 U.S.C. §371 and claims priority toInternational Application no. PCT/US2015/014692, filed Feb. 5, 2015,which claims the benefit of U.S. Provisional Application No. 61/936,143,filed Feb. 05, 2014 and PCT/US2015/014692 is a continuation-in-part ofU.S. application Ser. No. 14/486,636, filed Sep. 15, 2014 (now U.S. Pat.No. 9,119,832), each of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This invention provides for methods for treating mild brain injuries andother neurological disorders arising from such an injury in a subject byadministering to the subject an effective amount of a compositioncomprising ghrelin.

BACKGROUND OF THE INVENTION

Ghrelin, a 28-amino acid peptide predominantly secreted by gastricmucosa, is a neuroendocrine hormone that acts as an endogenous ligandfor growth hormone secretagogue receptor. Beyond the known effects onhunger regulation, ghrelin is known to have potent anti-inflammatoryproperties and has been shown to be protective in several models ofsevere neuronal injury.

Post traumatic brain injury (i.e., TBI) edema and the complicationsassociated with increased Intracranial pressure (ICP) account forapproximately 50% of death in hospitalized patients. Medically managingintracranial hypertension utilizes a strategy combining sedation andosmotic agents such as mannitol and hypertonic saline. Knowing thatghrelin has potent anti-inflammatory effects, we have shown that ghrelinprevents blood brain barrier (BBB) permeability, intestinal dysfunctionand systemic inflammation following TBI. The mechanism of these aboveobservations are unclear, but may be, at least in part, a result ofdecreasing transcription of inflammatory cytokines and end-cascadeeffects including decreasing apoptosis, and blood brain barrier leakage.Ghrelin treated TBI animals had a significant decrease in brainfluorescence correlating with decreased BBB vascular permeability. Wehave shown that ghrelin prevents post-TBI up-regulation of Aquaporin 4(AQP-4). Previous studies have shown that decreased expression of AQP-4was associated with prevention of BBB breakdown and brain edema.

Mild brain injuries (mBI), typically including concussions, having “yourbell rung”, and the like, describe an insult to the brain that, in turn,can cause long term injury to the brain. To date, there has been littleto no credible treatment of such mild brain injuries (mBI). Thus, thereis a significant unmet need for a therapy for treating mBI that does notimpose undesirable costs and delays and are effective.

SUMMARY OF THE INVENTION

This invention provides for methods of treating mild brain injury (mBI)in a subject having such an injury, comprising administering to thesubject an effective amount of ghrelin or ghrelin agonist, therebytreating the mBI. In some embodiments, ghrelin comprises a polypeptidecomprising at least one modification to the natural form of an aminoacid sequence ofGly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg(SEQ ID NO. 1). In some instances, the serine at amino acid position 2and/or serine at amino acid position 3 are/is acylated with an octanoylgroup (—C(O)(CH₂)₆CH₃. Amino acids at position 1 to 5 of SEQ ID NO. 1 ofthe ghrelin or ghrelin agonist can have a modification. In someembodiments, ghrelin increases uncoupling protein-2 (UCP-2) expression.In some embodiments, ghrelin increases UCP-2 expression in mitochondria.The amount delivered according to the methods herein, can be forexample, dosage of 2μg/kg per dose and/or per day. In an embodiment ofthe invention, ghrelin is des-acyl-ghrelin or ghrelin with nomodification of the primary amino acid sequence as provided in SEQ IDNO. 1. In another embodiment, ghrelin binds to a receptor other thanGHSR-la or ghrelin receptor, and wherein binding to a receptor otherthan GHSR-la or ghrelin receptor provides a therapeutic benefitfollowing mBI, for example, neuroprotection following mBI, repeated mBIor CTE. The therapeutic benefit such as neuroprotection following mBI orrepeated mBI may include reduced oxidative stress or reduced apoptosis.

In some embodiments, the mild brain injury comprises a concussion. Insome embodiments, the subject is a mammal. In some embodiments, thesubject is a human. In some embodiments, ghrelin is administered withinnot more than about 8, 24 or 72 hours after the mBI. In someembodiments, ghrelin is administered within not more than about 24 hoursafter the mBI. In some embodiments, ghrelin is administered within or atabout 0.1, 0.3, 0.5, 0.7, 1, 2, 3, 6, 8, 12, 18, 24, 36, 48, or 72 hoursafter the mBI.

This invention provides for methods of reducing the incidence orseverity of mBI in a subject, comprising administering to the subject aneffective amount of ghrelin, thereby reducing the incidence or severityof the mBI. In some embodiments, ghrelin is administered prior to anevent or activity with a potential for occurrence of mBI.

This invention provides for methods of reducing the amount of timerequired for recovery from a mild brain injury, comprising administeringto a patient suffering from a mild brain injury a therapeuticallyeffective amount of ghrelin within 72 hours of the mild brain injury. Insome embodiments, ghrelin is administered in a single dose. In someembodiments, ghrelin is administered at a dosage from 10 ng/kg per dayto 10 mg/kg per day.

Still further, ghrelin can be used in an assay to assess the ability ofcandidate compounds to effect increased uncoupling protein-2 (UCP-2)expression including increased UCP-2 expression in mitochondria. In suchassays, ghrelin is used as a control to determine the relative efficacyof the candidate compound or compounds. Suitable assays include, by wayof example only, competitive assays for binding of a candidate compoundor compounds to growth hormone secretagogue receptor 1a (i.e., GHSR) inthe presence of ghrelin as well as frontal affinity chromatography.

In yet another embodiment, a patient suffering loss of cognitive ormotor skills due to mBI and, in particular, repetitive mBI, can bemonitored for therapy or progression of such skills by correlating theghrelin level in the patient's brain over time. As the ghrelin levelsdecrease, there will be an increased need for intervention. Thisinvention also provides for methods of measuring ghrelin levels beforean activity, for example before the start of football, soccer, rugby orany other sport or activity season, and monitoring during the season toascertain if the player is at a level not qualified to play orparticipate by utilizing a test or an assay for measuring ghrelinlevels, such as a test or assay for determining levels in the blood.

In some embodiments, the present invention provides for a method fortreating a subject suffering from metabolic derangements associated withmBI or concussion, wherein such method comprises administration of aneffective amount of ghrelin to the subject, thereby treating the subjectsuffering from metabolic derangements associated with mBI or concussion.

In some embodiments, the present invention provides for a method fortreating a subject suffering from increased levels of reactive oxidativespecies (ROS) in neurons associated with mBI or concussion, wherein suchmethod comprises administration of an effective amount of ghrelin to thesubject so as to decrease levels of ROS in neurons associated with mBIor concussion, thereby treating the subject suffering from metabolicderangements associated with mBI or concussion.

In some embodiments, the present invention provides for a method forpreventing chronic traumatic encephalopathy (CTE) associated withrepeated mBI or concussions in a subject, wherein such method comprisesadministration of one or more doses of ghrelin to the subject, therebypreventing chronic traumatic encephalopathy (CTE) associated withrepeated mBI or concussions in a subject.

In some embodiments, the present invention provides for a method forpreventing damage to neurons associated with oxidative stress andoverproduction of reactive oxidative species (ROS) in a subject with oneor more incidence of mBI or concussion, wherein such method comprisesadministration of one or more doses of ghrelin to the subject, therebypreventing damage to neurons associated with oxidative stress andoverproduction of reactive oxidative species (ROS) in a subject with oneor more incidence of mBI or concussion.

In some embodiments, the present invention provides for a method fortreating injured or disrupted neuronal or axonal connection in the brainof a subject with one or more incidence of mBI, including concussion,wherein such method comprises administration of one or more doses ofghrelin to the subject, thereby treating injured or disrupted neuronalor axonal connection in the brain of a subject with one or moreincidence of mBI, including concussion. In one embodiment, the injuredor disrupted neuronal or axonal connection is mechanical disruption ofneuronal or axonal cytoskeleton. In another embodiment, the injured ordisrupted neuronal or axonal connection is disruption or alteredneuronal or axonal transport. In another embodiment, the injured ordisrupted neuronal or axonal connection is proteolysis, die-backdisconnection and reorganization. In one embodiment, treating injured ordisrupted neuronal or axonal connection in the brain of a subject withone or more incidence of mBI, including concussion, restores neuronal oraxonal connection in the brain of a subject following one or moreincidence of mBI, including concussion. In another embodiment, treatinginjured or disrupted neuronal or axonal connection in the brain of asubject with one or more incidence of mBI or concussion hastensrestoration of neuronal or axonal connection in the brain of a subjectfollowing one or more incidence of mBI, including concussion.

In some embodiments, the present invention provides for a method forpreventing or diminishing occurrence of an injured or disrupted neuronalor axonal connection in the brain of a subject with one or moreincidence of mBI, including concussion, wherein such method comprisesadministration of one or more doses of ghrelin to the subject, therebyreducing or preventing or diminishing occurrence of an injured ordisrupted neuronal or axonal connection in the brain of a subject withone or more incidence of mBI, including concussion. In one embodiment,the injured or disrupted neuronal or axonal connection is mechanicaldisruption of neuronal or axonal cytoskeleton. In another embodiment,the injured or disrupted neuronal or axonal connection is disruption oraltered neuronal or axonal transport. In another embodiment, the injuredor disrupted neuronal or axonal connection is proteolysis, die-backdisconnection and reorganization. In one embodiment, ghrelin isadministered prior to the occurrence of one or more incidence of mBI,including concussion. In another embodiment, ghrelin is administeredimmediately after the occurrence of an mBI, including concussion. Inanother embodiment, ghrelin is administered prior to and followingoccurrence of one or more mBI, including concussion. In anotherembodiment, multiple doses of ghrelin are administered.

In some embodiments, the present invention provides for a method fortreating perturbed brain physiology in a subject with one or moreincidence of mBI, including concussion, wherein such method comprisesadministration of one or more doses of ghrelin to the subject so as torestore normal neuronal depolarization, thereby treating perturbed brainphysiology in a subject with one or more incidence of mBI, includingconcussion. In one embodiment, treating perturbed brain physiology withghrelin restores normal brain physiology. In another embodiment,treating perturbed brain physiology with ghrelin hastens restoration ofnormal brain physiology.

In some embodiments, the present invention provides for a method forpreventing perturbed brain physiology in a subject with one or moreincidence of mBI, including concussion, wherein such method comprisesadministration of one or more doses of ghrelin to the subject so as tomaintain normal neuronal depolarization, thereby reducing or preventingperturbed brain physiology in a subject with one or more incidence ofmBI, including concussion. In one embodiment, ghrelin is administeredprior to the occurrence of one or more incidence of mBI, includingconcussion. In another embodiment, ghrelin is administered immediatelyafter the occurrence of an mBI, including concussion. In anotherembodiment, ghrelin is administered prior to and following occurrence ofone or more mBI, including concussion. In another embodiment, multipledoses of ghrelin are administered.

In some embodiments, the present invention provides for a method fortreating perturbed brain physiology in a subject with one or moreincidence of mBI or concussion, wherein such method comprisesadministration of one or more doses of ghrelin to the subject so as torestore normal release of excitatory neurotransmitters, thereby treatingperturbed brain physiology in a subject with one or more incidence ofmBI or concussion. In one embodiment, treating perturbed brainphysiology with ghrelin restores normal brain physiology. In anotherembodiment, treating perturbed brain physiology with ghrelin hastensrestoration of normal brain physiology.

In some embodiments, the present invention provides for a method forpreventing perturbed brain physiology in a subject with one or moreincidence of mBI or concussion, wherein such method comprisesadministration of one or more doses of ghrelin to the subject so as tomaintain normal release of excitatory neurotransmitters, therebyreducing or preventing perturbed brain physiology in a subject with oneor more incidence of mBI or concussion. In one embodiment, ghrelin isadministered prior to the occurrence of one or more incidence of mBI orconcussion. In another embodiment, ghrelin is administered immediatelyafter the occurrence of an mBI or concussion. In another embodiment,ghrelin is administered prior to and following occurrence of one or moremBI or concussion. In another embodiment, multiple doses of ghrelin areadministered.

In some embodiments, the present invention provides for a method fortreating perturbed brain physiology in a subject with one or moreincidence of mBI or concussion, wherein such method comprisesadministration of one or more doses of ghrelin to the subject so as torestore normal cerebral blood flow, thereby treating perturbed brainphysiology in a subject with one or more incidence of mBI or concussion.In one embodiment, treating perturbed brain physiology with ghrelinrestores normal brain physiology. In another embodiment, treatingperturbed brain physiology with ghrelin hastens restoration of normalbrain physiology.

In some embodiments, the present invention provides for a method forpreventing perturbed brain physiology in a subject with one or moreincidence of mBI or concussion, wherein such method comprisesadministration of one or more doses of ghrelin to the subject so as tomaintain normal cerebral blood flow, thereby reducing or preventingperturbed brain physiology in a subject with one or more incidence ofmBI or concussion. In one embodiment, ghrelin is administered prior tothe occurrence of one or more incidence of mBI or concussion. In anotherembodiment, ghrelin is administered immediately after the occurrence ofan mBI or concussion. In another embodiment, ghrelin is administeredprior to and following occurrence of one or more mBI or concussion. Inanother embodiment, multiple doses of ghrelin are administered.

In some embodiments, the present invention provides for a method fortreating perturbed brain physiology in a subject with one or moreincidence of mBI or concussion, wherein such method comprisesadministration of one or more doses of ghrelin to the subject so as torestore proper or normal axonal function, thereby treating perturbedbrain physiology in a subject with one or more incidence of mBI orconcussion. In one embodiment, treating perturbed brain physiology withghrelin restores normal brain physiology. In another embodiment,treating perturbed brain physiology with ghrelin hastens restoration ofnormal brain physiology.

In some embodiments, the present invention provides for a method forpreventing perturbed brain physiology in a subject with one or moreincidence of mBI or concussion, wherein such method comprisesadministration of one or more doses of ghrelin to the subject so as tomaintain proper or normal axonal function, thereby reducing orpreventing perturbed brain physiology in a subject with one or moreincidence of mBI or concussion. In one embodiment, ghrelin isadministered prior to the occurrence of one or more incidence of mBI orconcussion. In another embodiment, ghrelin is administered immediatelyafter the occurrence of an mBI or concussion. In another embodiment,ghrelin is administered prior to and following occurrence of one or moremBI or concussion. In another embodiment, multiple doses of ghrelin areadministered.

In some embodiments, the present invention provides for a method fortreating perturbed brain physiology in a subject with one or moreincidence of mBI or concussion, wherein such method comprisesadministration of one or more doses of ghrelin to the subject so as torestore brain metabolic function, thereby treating perturbed brainphysiology in a subject with one or more incidence of mBI or concussion.In one embodiment, treating perturbed brain physiology with ghrelinrestores normal brain physiology. In another embodiment, treatingperturbed brain physiology with ghrelin hastens restoration of normalbrain physiology.

In some embodiments, the present invention provides for a method forpreventing perturbed brain physiology in a subject with one or moreincidence of mBI or concussion, wherein such method comprisesadministration of one or more doses of ghrelin to the subject so as tomaintain brain metabolic function or balance, thereby reducing orpreventing perturbed brain physiology in a subject with one or moreincidence of mBI or concussion. In one embodiment, ghrelin isadministered prior to the occurrence of one or more incidence of mBI orconcussion. In another embodiment, ghrelin is administered immediatelyafter the occurrence of an mBI or concussion. In another embodiment,ghrelin is administered prior to and following occurrence of one or moremBI or concussion. In another embodiment, multiple doses of ghrelin areadministered.

In some embodiments, the present invention provides for a method fortreating perturbed brain physiology in a subject with one or moreincidence of mBI or concussion, wherein such method comprisesadministration of one or more doses of ghrelin to the subject so as torestore glucose metabolism, thereby treating perturbed brain physiologyin a subject with one or more incidence of mBI or concussion. In oneembodiment, treating perturbed brain physiology with ghrelin restoresnormal brain physiology. In another embodiment, treating perturbed brainphysiology with ghrelin hastens restoration of normal brain physiology.

In some embodiments, the present invention provides for a method forpreventing perturbed brain physiology in a subject with one or moreincidence of mBI or concussion, wherein such method comprisesadministration of one or more doses of ghrelin to the subject so as tomaintain glucose metabolism, thereby reducing or preventing perturbedbrain physiology in a subject with one or more incidence of mBI orconcussion. In one embodiment, ghrelin is administered prior to theoccurrence of one or more incidence of mBI or concussion. In anotherembodiment, ghrelin is administered immediately after the occurrence ofan mBI or concussion. In another embodiment, ghrelin is administeredprior to and following occurrence of one or more mBI or concussion. Inanother embodiment, multiple doses of ghrelin are administered.

In some embodiments, the present invention provides for a method forpreventing memory loss or headaches in a subject with mBI or concussion,wherein such method comprises administration of an effective amount ofghrelin to the subject in one or more doses, thereby preventing memoryloss and/or headaches in a subject with mBI or concussion.

Some embodiments relate to formulations for administration to a subject,which formulations can include a pharmaceutically acceptable carrier andghrelin having a carbon 14 (C14) content of less than 1 part pertrillion (ppt), wherein said formulation is suitable for delivery of aneffective amount of ghrelin to the brain of said patient so as to treatmild brain injuries. For example, any of the methods described above andelsewhere herein can utilize a ghrelin molecule have a C14 content ofless than 1 ppt.

Some embodiments relate to methods of monitoring a mild brain injury,the severity of an injury and/or the recovery from such an injury. Themethods can include, for example, administering a purified ghrelincompound, including for example, ghrelin with a C14 content of less than1 ppt, in a pharmaceutically acceptable composition to a subject thathas suffered a mild brain injury.

Some embodiments relate to methods of treating mild brain injury,reducing the incidence or severity of mBI in a subject, and/or reducingthe amount of time needed to recover from a mild brain injury. Themethods include providing or administering to a subject in need (e.g., asubject that has suffered, is at risk of suffering, is prone to suffer,and/or is about to participate in an activity with a high risk forsuffering, a mBI) an amount of ghrelin (including ghrelin with a C14content of less than 1 ppt) sufficient to provide a therapeuticallyeffective in vivo level of ghrelin to treat or reduce according to themethod, wherein the level is greater than the endogenous level ofghrelin in the subject. For example, the amount of administered ghrelincan be an amount sufficient to provide a blood level of ghrelin that isgreater than the usual or average endogenous blood level of ghrelin,such as 1.5, 2, 3, 5, 10, 20, 50, 100, 1,000 or up to 2,000 times thenormal endogenous blood level (or any sub value or sub range therebetween). In some case, the amount administered can result in a blood orplasma concentration of at least 55 picograms per milliliter. In someembodiments the greater ghrelin levels can be achieved within hours ofthe injury (e.g., less than 8 hours after the injury). They also can bemaintained above endogenous levels for some period of time that issufficient to provide the desired therapeutic benefit, for example, forat least 30 minutes to 24 hours (or any sub value or sub range therebetween). Endogenous ghrelin levels are not sufficient for treating mBIor reducing the incidence, severity or the time needed to recover asreadily evidenced by the long term damage done to the brain byrepetitive concussive injuries (mBI). The instant embodiments provide abenefit and result that do not occur naturally in the body withendogenous levels. Such a benefit was unknown prior to the instantembodiments associated with the invention. In some embodiments, themethods can further include selecting or identifying a subject that hassuffered, is at risk of suffering, is prone to suffer, and/or is aboutto participate in an activity with a high risk for suffering, a mBI,prior to administration of the ghrelin.

Some embodiments relate to methods for determining the efficacy of acompound treating a patient suffering mild brain injury (mBI). Themethods can include, for example, i) determining the expression level ofuncoupling protein-2 (UCP-2) in a biological sample obtained from thepatient treated with the compound; ii) comparing the expression level ofUCP-2 to a biological sample obtained from a subject treated withghrelin (as described herein including with the modifications describedherein); and iii) determining the efficacy of the compound, wherein thecompound is efficacious when the expression level of UCP-2 induced bythe compound is equal to (within at least 10%) or greater than theexpression level of UCP-2 induced by ghrelin.

Some embodiments relate to methods for treating a patient suffering lossof cognitive or motor skills due to mild brain injury (mBI). The methodscan include, for example, i) determining the level of ghrelin in thepatient's brain over a period of time; ii) administering ghrelin(including the modified and C14 versions described herein) to thepatient; and iii) periodically repeating step ii) during treatment whenthe level of ghrelin falls below a normal range as a basis to determinethe efficacy of the treatment, wherein an increase in the level ofghrelin in the brain demonstrates an improvement in the patient'scognitive or motor skill condition.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments of this disclosure are drawings which illustrate byexemplification only, and not limitation, wherein:

FIG. 1 depicts principle of assaying oxidative burst within inflammatorycells. Dihydrorhodamine 123 (DHR 123) diffuses across the cell membrane.When it encounters reactive oxygen species (ROS), DHR 123 is oxidizedand fluoresces green. The fluorescence is then measured and presented asarbitrary fluorescence unit (AFU), wherein higher intensity meansgreater oxidative burst and, therefore greater concentration of amountof ROS (See Chen, et al., “Measurement of oxidative burst inneutrophils,” Methods Mol Biol., 844: 115-124 (2012); which isincorporated herein by reference in its entirety).

FIG. 2 depicts subcutaneous administration of ghrelin resulting inreduced oxidative burst in inflammatory cells following mBI. Rightwardshift in peak reflects higher oxidative burst. Higher oxidative burst isobserved in mBI alone, and lower oxidative burst is observed in ghrelintreated mBI. For the ghrelin treatment, the peak is shifted left in linewith control meaning lower oxidative burst.

FIG. 3 depicts oxidative burst of ipsilateral whole brain following mildBI. Mild BI plus ghrelin reduces oxidative burst as quantified byarbitrary fluorescence unit (AFU). Oxidative burst is increased inanimals following mBI compared to sham control (7963 AFU±2900 (mild BI)vs. 4624 AFU±1858 (sham); n=5 animals in each group). Ghrelin treatmentreduces the oxidative burst compared to mBI (7963 AFU±2900 (mild BI) vs.3257 AFU±1031 (mild BI+ghrelin (2 doses)); p-value of 0.048 usingStudent's t-test). AFU equals mean fluorescence intensity.

Some or all of the figures are schematic representations forexemplification; hence, they do not necessarily depict the actualrelative sizes or locations of the elements shown. The figures arepresented for the purpose of illustrating one or more embodiments withthe explicit understanding that they will not be used to limit the scopeor the meaning of the claims that follow below.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the present disclosure is not limited toparticular embodiments described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present disclosure will belimited only by the appended claims.

The detailed description of the present disclosure is divided intovarious sections only for the reader's convenience and disclosure foundin any section may be combined with that in another section. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which the present disclosure belongs.

Definitions

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acompound” includes a plurality of compounds.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the present disclosure belongs. As used herein thefollowing terms have the following meanings.

As used herein, the term “about” when used before a numericaldesignation, e.g., temperature, time, amount, concentration, and suchother, including a range, indicates approximations which may vary by (+)or (−) 10%, 5% or 1%.

As used herein, the term “administration” can be effected in one dose,continuously or intermittently or by several subdoses which in theaggregate provide for a single dose. Dosing can be conducted throughoutthe course of treatment. Methods of determining the most effective meansand dosage of administration are known to those of skill in the art andwill vary with the composition used for therapy, the purpose of thetherapy, the target cell being treated and the subject being treated.Single or multiple administrations can be carried out with the doselevel and pattern being selected by the treating physician. Suitabledosage formulations and methods of administering the agents are known inthe art. Route of administration can also be determined and method ofdetermining the most effective route of administration are known tothose of skill in the art and will vary with the composition used fortreatment, the purpose of the treatment, the health condition or diseasestage of the subject being treated and target cell or tissue.Non-limiting examples of route of administration include oraladministration, vaginal, nasal administration, injection, topicalapplication, sublingual, pulmonary, and by suppository.

As used herein, the term “affinity” refers to the strength of bindingbetween receptors and their ligands, for example, between an antibodyand its antigen.

As used herein, the term “amino acid residue” refers to an amino acidformed upon chemical digestion (hydrolysis) of a polypeptide at itspeptide linkages. Unless otherwise specified, the amino acid encompassesL-amino acid, including both natural amino acid and synthetic amino acidor the like as long as the desired functional property is retained bythe polypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. Standard polypeptideabbreviations for amino acid residues are as follows: A (Ala orAlanine); C (Cys or Cysteine); D (Asp or Aspartic Acid); E (Glu orGlutamic Acid); F (Phe or Phenylalanine); G (Gly or Glycine); H (His orHistidine); I (Ile or Isoleucine); K (Lys or Lysine); L (Leu orLeucine); M (Met or Methionine); N (Asn or Asparagine); P (Pro orProline); Q (Gln or Glutamine); R (Arg or Arginine); S (Ser or Serine);T (Thr or Threonine); V (Val or Valine); W (Trp or Tryptophan); X (Xaaor Unknown or Other); Y (Tyr or Tyrosine); and Z (Glx/Gln/Glu orGlutamic Acid/Glutamine). All amino acid residue sequences representedherein by formula have a left-to-right orientation in the conventionaldirection of amino terminus to carboxy terminus. The phrase “amino acidresidue” is broadly defined to include the naturally occurring andmodified and non-naturally occurring amino acids. A dash at thebeginning or end of an amino acid residue sequence indicates a peptidebond to a further sequence of one or more amino acid residues or acovalent bond to an amino-terminal group such as NH₂ or acetyl or to acarboxy-terminal group such as COOH.

As used herein, the terms “comprising” or “comprises” is intended tomean that the compositions and methods include the recited elements, butnot excluding others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the stated purpose. Thus,a composition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) of the present disclosure.“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps. Embodiments defined by each ofthese transition terms are within the scope of the present disclosure.

As used herein, the term “ghrelin” is a polypeptide that may have 28amino acid sequence as set forth in SEQ ID No. 1, and can include theoctanoyl acylation as described above. Human ghrelin may be apolypeptide having the amino acid sequence as set forth in GenBank®Accession No. NP_057446 or Swiss-Prot Identifier GHRL_HUMAN. Humanghrelin preprotein has 117 amino acids. This preprotein may undergo thefollowing post-translational processing. The signal peptide (amino acids1-23) is removed and the remaining 94 amino acids are cleaved by aprotease to provide a mature 28 amino acid ghrelin (amino acids 24-51)or a mature 27 amino acid ghrelin (amino acids 24-50) and a mature 23amino acid obestatin (amino acids 76-98). In another embodiment, ghrelincomprises a polypeptide comprising at least one modification to thenatural form of an amino acid sequence ofGly-Ser-Ser-Phe-Leu-Ser-Pro-Ser-Gln-Lys-Pro-Gln-Asn-Lys-Val-Lys-Ser-Ser-Arg-Ile(e.g., SEQ ID NO. 4). In another embodiment, ghrelin comprises apolypeptide comprising at least one modification to the natural form ofan amino acid sequence ofGly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Lys-Ala-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg(SEQ ID NO. 5). In another embodiment, ghrelin comprises a polypeptidecomprising at least one modification to the natural form of an aminoacid sequence ofGly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Lys-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Ala-Ala-Lys-Leu-Lys-Pro-Arg(SEQ ID NO. 6). In another embodiment, ghrelin comprises a polypeptidecomprising at least one modification to the natural form of an aminoacid sequence ofGly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Ala-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg(SEQ ID NO. 7). In another embodiment, ghrelin comprises a polypeptidecomprising at least one modification to the natural form of an aminoacid sequence ofGly-Ser-Ser-Phe-Leu-Ser-Pro-Thr-Tyr-Lys-Asn-Ile-Gln-Gln-Gln-Lys-Asp-Thr-Arg-Lys-Pro-Thr-Ala-Arg-Leu-His(SEQ ID NO. 8). In yet another embodiment, ghrelin comprises apolypeptide comprising at least one modification to the natural form ofan amino acid sequence ofGly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Lys-Leu-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg(SEQ ID NO. 9). Modifications to ghrelin may include adding or removingamino acids so long as the ghrelin so modified retains its therapeuticvalue (e.g., by adding or removing any of 1, 2, 3, 4 or 5 amino acidsfrom the molecule).

Some embodiments relate to and can utilize ghrelin molecules that have acarbon 14 (C14) content less than found in endogenously produced ghrelinmolecules or in ghrelin that has a C14 content about the same asatmospheric C14 levels. For example, ghrelin molecules can have at leastone carbon atom or carbon containing moiety that is from fossil derivedreagents that have a C14 content less than found in endogenous moleculesor less than atmospheric levels. In some embodiments, the ghrelinmolecules can have all, substantially all or at least a some carbonhaving a C14 content less than found endogenously or less thanatmospheric levels. For example, one or more of the amino acids of asequence can include carbon and have a C14 content less than found inendogenous amino acids or less than atmospheric levels. In other casesan entire sequence can include carbon and have a C14 content less thanfound endogenously or less than atmospheric levels. Still, in otherembodiments, a ghrelin molecule can be modified, for example to have anoctanoyl or other like group, and that octanoyl group can have a C14content less than endogenous ghrelin C14 levels or less than atmosphericlevels. Further examples and embodiments are described below andelsewhere herein.

In some embodiments, ghrelin molecule can have a C14 content of lessthan 0.9 ppt, 0.95 ppt, 1.05 ppt, 1.10 ppt, 1.15 ppt, 1.2 ppt oratmospheric content of C14. In some embodiments, ghrelin molecule canhave a C14 content that is from about 1% to 50% (or any value or subrange therein) less than the content of C14 in endogenous ghrelin or thecontent of atmospheric C14. For example, a molecule according to someembodiments can have about 5% to about 11% less C14 content. Ghrelinwith C14 content of less than 0.9 ppt, 0.95 ppt, 1.0 ppt, 1.05 ppt, 1.10ppt, 1.15 ppt, 1.2 ppt or atmospheric content of C14, or with a lesserpercentage of C14 as discussed herein, may be obtained by peptide orchemical synthesis using reactants with carbons free of C14, less than 1ppt C14 or deficient in C14 relative to the atmospheric content of C14.Alternatively, ghrelin with C14 content of less than 0.9 ppt, 0.95 ppt,1.0 ppt, 1.05 ppt, 1.10 ppt, 1.15 ppt, 1.2 ppt or atmospheric content ofC14 may be produced in vitro by enzymatic methods using startingmaterials with a carbon content free of C14, substantially free of C14,less than 1 ppt C14 or deficient in C14 relative to the atmosphericcontent of C14. Such enzymatic methods may include cell-free proteinsynthesis system or coupled in vitro transcription-translation systembased on cellular extracts prepared from bacteria, yeast, wheat germ,insect and/or mammalian cells using aminoacyl-tRNAs charged with aminoacids with a carbon content free of C14, substantially free of C14, lessthan 1 ppt C14 or deficient in C14 relative to the atmospheric contentof C14. In an alternative method, ghrelin with C14 content of less than0.9 ppt, 0.95 ppt, 1.0 ppt, 1.05 ppt, 1.10 ppt, 1.15 ppt, 1.2 ppt oratmospheric content of C14 may be produced by recombinant methods inbacterial, yeast, insect and/or mammalian cells following introductionof an expression system with a cDNA comprising ghrelin-encoded sequencesand culturing the cells in a medium with a carbon content free of C14,substantially free of C14, less than 1 ppt C14 or deficient in C14relative to the atmospheric content of C14. Alternatively, the mediummay include glucose, galactose, sugars, glycerol, pyruvate, acetates,metabolites, fatty acids, and/or amino acids with a carbon content freeof C14, substantially free of C14, less than 1 ppt C14 or deficient inC14 relative to the atmospheric content of C14. Methods for changingstable isotopic content of proteins may be found in Becker et al., 2008(G. W. Becker (2008) Stable isotopic labeling of proteins forquantitative proteomic applications. Briefings in Functional GenomicsProteins 7 (5): 371-382, which is incorporated herein by reference inits entirety) Ghrelin may be co-expressed with or exposed to ghrelinO-acyl transferase (GOAT) to permit fatty acid modification of theprimary sequence of ghrelin at serine at amino acid position 3 so as toproduce a biologically active ghrelin capable of being bound andactivating the ghrelin receptor (GHSR-la or growth hormone secretagoguereceptor type 1a). The modification may be an octanoic acid modificationof ghrelin so as to produce octanoyl-ghrelin with a carbon content freeof C14, substantially free of C14, less than 1 ppt C14 or deficient inC14 relative to the atmospheric content of C14.

In some embodiments, ghrelin with C14 content of less than 0.9 ppt, 0.95ppt, 1.0 ppt, 1.05 ppt, 1.10 ppt, 1.15 ppt, 1.2 ppt or atmosphericcontent of C14 (or having a percentage as discussed herein) may beobtained following modification of the primary sequence of ghrelin (SEQID NO. 1) with a fatty acid with a carbon content free of C14,substantially free of C14, less than 1 ppt C14 or deficient in C14relative to the atmospheric content of C14. Such fatty acids may bechemically synthesized with a carbon content free of C14, substantiallyfree of C14, less than 1 ppt C14 or deficient in C14 relative to theatmospheric content of C14 or produced in a cell cultured in a mediumwherein carbon source used to synthesize the fatty acid or fatty acidsis free of C14, substantially free of C14, less than 1 ppt C14 ordeficient in C14 relative to the atmospheric content of C14. In someembodiments, the fatty acid or fatty acids are conjugated to coenzyme A(CoA) and the fatty acid in the resulting fatty acid-CoA thioesters istransferred to serine at amino acid position 3 of ghrelin by ghrelinO-acyl transferase (GOAT), so as to produce a fatty acid-modifiedghrelin with a carbon content free of C14, substantially free of C14,less than 1 ppt C14 or deficient in C14 relative to the atmosphericcontent of C14. In some embodiments, fatty acids are straight chainfatty acids with a carbon content of C2, C3, C4, C5, C6, C7, C8, C9,C10, C11, C12, C13, C14, C15, C16, C17, C18, C19 or C20 and having ageneral chemical formula of (CH)₃—(CH₂)_(n−2)—COOH, wherein “n” is thenumber of carbons in the fatty acid. In a preferred embodiment, thefatty acid is a C8 octanoic acid or C14 tetradecanoic acid.

In a more preferred embodiment, the fatty acid is octanoic acid and thefatty acid-modified ghrelin is octanoyl-modified ghrelin at serine aminoacid position 3.

In some embodiments, fatty acid or fatty acids may be conjugated toghrelin at serine amino acid position 3. In some embodiments, fatty acidor fatty acids may be conjugated to ghrelin at a position other thanserine amino acid position 3. In some embodiments, fatty acid or fattyacids may be conjugated to ghrelin at serine amino acid position 2. Insome embodiments, fatty acid or fatty acids may be conjugated to ghrelinat serine amino acid position 2 and serine amino acid position 3. Insome embodiments, fatty acid or fatty acids may be conjugated to ghrelinat one or more amino acids.

In some embodiments, fatty acid or fatty acids may be conjugated toimmature ghrelin (such as preproghrelin or proghrelin) and then fattyacid- or fatty acids-modified ghrelin is processed to a mature ghrelinthat can activate the ghrelin receptor (GHSR-1a). Processing of immatureghrelin may be in vitro or in vivo and may be carried out by proteolyticenzymes. In some embodiments, fatty acid or fatty acids may beconjugated to a mature ghrelin having the amino acid sequence asprovided in SEQ ID NO. 1.

In some embodiments, ghrelin with one or more modifications is anisolated ghrelin with one or more modifications. In a preferredembodiment, ghrelin with one or more modifications is an isolatedghrelin with one or more fatty acid modifications. In a more preferredembodiment, ghrelin with one or more modifications is an isolatedghrelin acylated at serine 3 with octanoic acid, such as an isolatedoctanoyl-ghrelin.

C14-deficient starting material used in the synthesis of ghrelin with acarbon content free of C14, substantially free of C14, less than 1 pptC14 or deficient in C14 relative to the atmospheric content of C14 maybe obtained from carbon sources not participating in atmospheric carboncycle or by fractionating naturally occurring carbon isotope to obtaincarbons free of C14, substantially free of C14, less than 1 ppt C14 ordeficient in C14 relative to the atmospheric content of C14. Suchcarbons will be enriched in carbon-12 (C12) and/or carbon-13 (C13) anddepleted of C14. Methods for isotope fractionation, enrichment ordepletion are known in the art and may be based on diffusion,centrifugation, electromagnetism, laser excitation, kinetic isotopeeffect, chemical methods, gravity, evaporation, and cryogenicdistillation among many other methods of isotope fractionation.

The ghrelin as used in the various embodiments described herein can beany suitable ghrelin, including for example, ghrelin that is producedusing recombinant methods and ghrelin that is chemically synthesized.Regardless of the source, the ghrelin can be used and administered asdescribed herein. The ghrelin can be isolated ghrelin, for example,ghrelin obtained following fractionation or separation of ghrelin awayfrom other constituents present in a sample in which ghrelin is producedor synthesized as well as modified. The isolated ghrelin need not bepure ghrelin. While it is desirable to have pure ghrelin or essentiallypure ghrelin, isolated ghrelin may be used without achieving absolutepurity so long as ghrelin has been fractionated or separated away fromconstituents whose presence is not desirable in a preparation of theisolated ghrelin. It is noted that following fractionation of ghrelin,the ghrelin may be introduced into solution or used as a mixture oradmixture in the presence of other components such as pharmaceuticalcarrier(s), lubricant(s), preservative(s), buffer(s), salt(s), sugar(s),colorant(s), pharmaceutically acceptable excipient(s) and combinationthereof.

As used herein, the term “ghrelin receptor” refers to any naturallyoccurring molecule to which ghrelin binds and induces a biologicalactivity. Ghrelin is known to bind to growth hormone secretagoguereceptor 1a (i.e., GHSR), however, the present disclosure is not limitedto a specific type of receptor.

As used herein, the term “individual” or “subject” is an animal or humansusceptible to a condition, in particular mBI or concussion. In someembodiments, the individual is a mammal, including human and non-humanmammals such as dogs, cats, pigs, cows, sheep, goats, horses, rats, andmice.

As used herein, the term “mild brain injury” (mBI) sometimes referred asa “mild traumatic brain injury” (mTBI) refers to a non-disease eventcommonly caused by an injury resulting in an insult to the brain. mBImay be caused, for example, by impact forces, in which the head strikesor is struck by something, or impulsive forces, in which the head moveswithout itself being subject to blunt trauma (for example, when thechest hits something and the head snaps forward; as a result of rapidacceleration or deceleration of the head; acceleration or decelerationof the brain of a subject producing a change in a qualitative orquantifiable clinical parameter related to functional well-being of thesubject; or differential acceleration or deceleration of the brain ofthe subject relative to the skull of the subject wherein differentialacceleration or deceleration produces a change in a qualitative orquantifiable clinical parameter related to functional well-being of thesubject). mBI commonly results, for example, from a sports-relatedinjury, a motor vehicle accident, an accidental fall, or an assault.Although the vast majority of such injuries improve through naturalrecovery the damage caused by such an injury or repetitive injuries cancause long term deficits in cognitive and/or motor skill functions.

Mild BI is different from and has a distinct pathology as compared todiseases such as acute traumatic brain insults such as strokes (ischemicor hemorrhagic), AVM's, brain tumors, and the like.

Moreover, mBI is distinct from severe TBI. Mild brain injury and severetraumatic brain injury are dramatically different injuries. Mild BI andsevere TBI have different characteristic and symptoms, differentmortality rates, different treatment protocols, etc. Severe TBI is aninjury state where there is an unacceptable level of mortality andpermanent disability in humans within hours or days of the injury.According to the Centers for Disease Control, such injuries are acontributing factor to a third (30%) of all injury-related deaths in theUnited States (See, National Vital Statistics System (NVSS), 2006-2010).Severe TBIs often are caused, for example, by automobile accidents andcombat gunshot and bomb wounds to the head. Severe TBI typically hasdirect and overt brain hemorrhage, brain cell destruction, severe brainedema and severe cellular apoptosis, all characteristic of anatomicalchanges or anatomical injury to the brain of the injured patient—all ofwhich can be detected by non-invasive anatomical imaging methods. Due tothe severity of such symptoms and risk of imminent death and disability,operative intervention and critical care are paramount. Severe TBIresults in adverse anatomical changes in the patient's brain (observableat a gross anatomy level) resulting in immediate and permanent loss ofbrain function, if not death.

On the other hand, mild brain injury (mBI) causes changes in the brainfunction at a metabolic/molecular level in the affected neurons. Assuch, it does not typically cause mortality or permanent disability inthe time frame provided above and patients often resume normalactivities of daily living and functionality within hours or days of themBI.

The body's response to severe TBI consists of a large number ofdiffering responses including those initiated by hypoxia, blood loss andsignificant neuron death, to name just a few. The body's response to mBIis significantly different as hemorrhaging and blood loss, tissue loss,hypoxia, significant neuron death etc. are not part of themanifestations of this condition.

Consequently, therapeutic treatments for each injury are significantlydifferent. The same therapeutic methodology used for severe TBI, forexample, surgery, deep sedation, mannitol therapy and operativedecompression, would be contra-indicated for treatment of mBI, forexample, concussion, which is a type of mild brain injury. The inverse,such as rest and mental relaxation with little intervention forrecovering from mBI, is likewise true.

As used herein, the term “polypeptide” or “peptide” is intended toencompass a singular “polypeptide” as well as plural “polypeptides,” andrefers to a molecule composed of monomers (amino acids) linearly linkedby amide bonds (also known as peptide bonds). The term “polypeptide”refers to any chain or chains of two or more amino acids, and does notrefer to a specific length of the product. Thus, peptides, dipeptides,tripeptides, oligopeptides, “protein,” “amino acid chain,” or any otherterm used to refer to a chain or chains of two or more amino acids, areincluded within the definition of “polypeptide,” and the term“polypeptide” may be used instead of, or interchangeably with any ofthese terms. The term “polypeptide” is also intended to refer to theproducts of post-expression modifications of the polypeptide, includingwithout limitation glycosylation, acetylation, phosphorylation,amidation, acylation, acylation by fatty acid, fatty acid-modification,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids.Modification may be fatty acid modification or triglyceridemodification. Fatty acid modification may be a short to medium-chainfatty acid. The short fatty acid may be a two-carbon fatty acid oracetic acid. Medium chain fatty acid may be 14-carbon fatty acid ortetradecanoic acid. Modification with a fatty acid may be acylation ofSEQ ID NO. 1 at serine amino acid position 2 and/or serine amino acidposition 3. Modification may be catalyzed by ghrelin O-acyl transferase(GOAT) of fatty acid thioester and ghrelin as substrates. In oneembodiment, post-translationally modified ghrelin may be bound and/orrecognized by growth hormone secretagogue receptor type 1a (GHSR-1a) orghrelin receptor. In one embodiment, post-translationally modifiedghrelin may be fatty acid-acylated ghrelin at serine amino acid position2 and/or serine amino acid position 3 bound and/or recognized by GHSR-laor ghrelin receptor. A polypeptide may be derived from a naturalbiological source or produced by recombinant technology, but is notnecessarily translated from a designated nucleic acid sequence. It maybe generated in any manner, including by chemical synthesis. The term“Polypeptide” or “Peptide” also refers to a compound comprising aplurality of amino acids linked therein via peptide linkages. Here, theamino acid (also called an amino acid residue) includes naturallyoccurring amino acids represented by formula: NH₂—CH(R′)—COOH, whereinR′ is a naturally occurring substituent group, as well as its D,L-optical isomers etc. There is also a peptide, wherein a certainnaturally occurring amino acid is replaced by a modified amino acid. Themodified amino acid includes the amino acids of the above formulawherein the substituent group R′ is further modified, its D, L-opticalisomers thereof, and non-natural amino acids wherein e.g. varioussubstituent groups are bound to the substituent group R′ of the aboveformula via or not via an ester, ether, thioester, thioether, amide,carbamide or thiocarbamide linkage. The modified amino acid alsoincludes non-natural amino acids whose amino groups are replaced bylower alkyl groups.

As used herein, the term “secretagogue” is a substance stimulatinggrowth hormone release, such as ghrelin. In some embodiments, theghrelin binds to the growth hormone secretagogue receptor GHS-R 1a(GHSR). The ghrelin compounds described herein are active at thereceptor for growth hormone secretagogue (GHS), e.g., the receptor GHS-R1a. The compounds can bind to GHS-R 1a, and stimulate receptor activity.In some embodiments, the compounds can bind other receptors and,optionally, stimulate their activity. In some embodiments, ghrelinincreases uncoupling protein-2 (UCP-2) expression. In some embodiments,ghrelin increases UCP-2 expression in mitochondria. In some embodiments,ghrelin prevents the metabolic consequence of mBI and any associatedchronic conditions.

Treatment of Mild Brain Injury (mBI) and Other Neurological Disorders

The present disclosure is directed to the identification of a novel usefor ghrelin in mBI. Mild BI, including concussion, has a significantlydifferent pathology than other traumas associated with brain disease andillness and severe traumatic brain injuries (severe TBI) such as thosecausing ischemia. Mild BI does not cause the massive tissue and cellulardamage as observed in severe traumatic brain disease. Rather mBI causessubtle metabolic changes within the brain, specifically oxidative stressand overproduction of reactive oxygen species (ROS) which, in turn, candamage neuroconnectivity and lead to neuron damage and encephalopathicand psychological changes with recurrent injury.

The present disclosure utilizes ghrelin in treating mBI. Unlike severetraumatic injury to the brain, mBI does not show acute neuronalhistological changes, severe neuronal inflammation or significantcellular or vasogenic edema (i.e., blood brain barrier breakdown).Rather, mBI is a clinical condition generally associated with a list ofclinical symptoms. It sometimes is associated with loss of consciousnessbut does not necessarily show significant radiological changes. Symptomsthat often are reported include, without limitation, headache, “pressurein head,” neck pain, nausea or vomiting, dizziness, blurred vision,balance problems, sensitivity to light, sensitivity to noise, feelingslowed down, feeling like “in a fog,” “don't feel right,” difficultyconcentrating, difficulty remembering, fatigue or low energy, confusion,drowsiness, trouble falling asleep, more emotional, irritability,sadness and being nervous or anxious. In some non-limiting embodimentsthe methods herein can include or relate to treating or reducing one ormore of the above listed symptoms. Most importantly, repeatedconcussions, as observed in the military and in sports such as footballand hockey, cause a series of metabolic disturbances and the formationof ROS. These series of events lead to chronic traumatic encephalopathy(CTE) as witnessed in the autopsies of several deceased professionalfootball players.

In some embodiments, ghrelin has the ability to decrease ROS and thesubsequent damage caused to neurons following mBI. This is especiallyimportant for chronic concussions where preventing the inflammatory,excitatory milieu of ROS would have significant clinical impact. Ghrelintreatments decrease ROS, and therefore prevent the metabolic consequenceof concussions and the chronic conditions that are associated thereof.

In some embodiments, the therapeutic effect of ghrelin in mBI iscompared to its therapeutic effect in severe TBI. The etiology, imaging,and assessment of mBI lack a cohesive explanation for the observedcognitive deficits of chronic headaches, memory loss and sleepimpairment. Only the most advanced and cumbersome technology can evendetect subtle changes associated with mBI. Therefore, unlike severe TBI,where a definitive mass lesion is identified with anatomic and cellularchanges, mBI is undetectable radiologically, nevertheless, specificmetabolic derangements occur. The present disclosure describes thatghrelin can be potent neuro-conservative agents in mBI.

Without being limited by any theory, the present disclosure alsodescribes the biological function of ghrelin following mBI. After mBI,the metabolic needs of the cell, including glucose requirements,increase. Mild injured cells have significant metabolic derangementscausing reactive oxygen species on neurons. This metabolic stress andincreased metabolic needs is the fundamental concept underlying acuteconcussion management. Decreasing ROS and improving glucose uptake ofthe brain or neuronal cells and axons can restore intracellular functionand remain viable. This prevents or reduces concussion-induceddysfunction and overall improves neurocognitive outcome, as well asprevents memory loss, and chronic mBI states of headaches anddevelopment of chronic traumatic encephalopathy.

The present disclosure provides for a method of treating mild braininjury (mild BI or mBI) in a subject, comprising administering to thesubject (e.g., a subject that has a mBI) an effective amount of acompound comprising ghrelin, thereby treating the mBI. The ghrelin canbe administered for the purpose of treating the mBI in a therapeuticallyeffective amount for the mBI. In some embodiments, the methods canfurther include selecting or identifying a subject that has suffered, isat risk of suffering, is prone to suffer, and/or is about to participatein an activity with a high risk for suffering, a mBI, prior toadministration of the ghrelin.

In some embodiments, the mild brain injury comprises a concussion. Insome embodiments, the subject that undergoes the method of treatment isa mammal. In some embodiments, the subject is a human, monkey, cow,goat, sheep, mouse, rat, cat, dog, horse, hamster, pig, fish andchicken. In some embodiments, ghrelin is administered within about 72hours of the mBI. In some embodiments, ghrelin is administered withinabout 24 hours of the mBI. In some embodiments, ghrelin is administeredat about 0.1, 0.3, 0.5, 0.7, 1, 2, 3, 6, 12, 18, 24, 36, 48, or 72 hoursafter the mBI.

The present disclosure also provides a method of treating mild braininjury (mBI) in a subject, comprising administering to the subject atherapeutically effective amount of ghrelin, thereby treating the mBI,wherein the ghrelin is the sole active agent. In some embodiments, theghrelin has the sequence ofGly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg(SEQ ID NO. 1). In some embodiments, the ghrelin is modified with one ormore fatty acids. In some embodiments, the fatty acid is an octanoicacid. In some embodiments, the ghrelin is modified at serine at aminoacid position 2 and/or serine at amino acid position 3 of SEQ ID NO. 1.In some embodiments, the ghrelin is modified at serine at amino acidposition 2 and/or serine amino acid position 3 with an octanoic acid. Insome embodiments, modification of ghrelin is acylation of ghrelin atserine at amino acid position 2 and/or serine at amino acid position 3with an octanoic acid. In some embodiments, the ghrelin so modified isan octanoyl-ghrelin with an acylation at serine at amino acid position 3by the octanoic acid. In some embodiments, the mBI comprises aconcussion. In some embodiments, the subject is a mammal. In someembodiments, ghrelin is administered, at least for a firstadministration (but not limited to), within or in less than about 24hours, or less than 48 hours, or less than 72 hours of the mBI, or anysub value or subrange between immediately after the injury and thosetime periods. In some embodiments, ghrelin is administered within notmore than about 8 hours of the mBI. In some embodiments the ghrelin canbe administered with about 6 hours of the mBI. In some embodiments, theghrelin can be administered a single dose, or in multiple doses, forexample 2-3 doses. The multiple doses can have at least a first doseadministered with the time period mentioned herein and one or moresubsequent doses after the time period. In some embodiments, the ghrelinis administered in an amount that results in ghrelin blood levels beingat least 1, at least 1.5, at least two times (or more) greater than theendogenous ghrelin blood levels of the subject. In some embodiments,ghrelin is administered in a single dose. In some embodiments, ghrelinis administered at a dosage from 10 ng/kg per day to 10 mg/kg per day orper dose. In some embodiments, the dosage of ghrelin can be between 1microgram and 15 micrograms per kg, for example about 10 μg/kg. In someembodiments, ghrelin is administered at a dosage of 2μg/kg per dose orin some case per day. In some embodiments, the ghrelin has a carbon 14(C14) content of less than 1 part per trillion (ppt).

The present disclosure also provides a method of treating mild braininjury (mBI) in a subject, comprising administering to the subject atherapeutically effective amount of ghrelin in an amount that providesblood levels of ghrelin that are at least 1.5 times greater thanendogenous ghrelin blood levels of the subject, thereby treating themBI. The ghrelin can be the sole active agent. In some embodiments, theghrelin has the sequence ofGly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg(SEQ ID NO. 1). In some embodiments, the ghrelin is modified with one ormore fatty acids. In some embodiments, the fatty acid is an octanoicacid. In some embodiments, the ghrelin is modified at serine at aminoacid position 2 and/or serine at amino acid position 3 of SEQ ID NO. 1.In some embodiments, the ghrelin is modified at serine at amino acidposition 2 and/or serine at amino acid position 3 with an octanoic acid.In some embodiments, modification of ghrelin is acylation of ghrelin atserine at amino acid position 2 and/or serine at amino acid position 3with an octanoic acid. In some embodiments, the ghrelin so modified isan octanoyl-ghrelin with an acylation at serine at amino acid position 3by the octanoic acid. In some embodiments, the mBI comprises aconcussion. In some embodiments, ghrelin is administered in a singledose. In some embodiments, the amount administered provides a bloodlevel of at least 1.0 to 100 times greater the amount found endogenouslyin the subject. In some embodiments, the amount administered results ina blood level of at least 55 picograms/mL. In some embodiments, ghrelinis administered at a dosage from 10 ng/kg per day to 10 mg/kg per dosageand/or per day. In some embodiments, the dosage of ghrelin can bebetween 1 microgram and 15 micrograms per kg, for example about 10μg/kg. In some embodiments, ghrelin is administered at a dosage of2μg/kg per dose or in some case per day. In some embodiments, theghrelin has a carbon 14 (C14) content of less than 1 part per trillion(ppt). In some embodiments, the ghrelin is administered in an amountthat provides blood levels of ghrelin that are at least 1.0, 2.0 times,or more, greater than endogenous ghrelin blood levels of the subject,thereby treating the mBI.

The present disclosure provides a method of treating mild brain injury(mBI) in a subject, comprising administering to the subject atherapeutically effective amount of a pharmaceutical compositionconsisting essentially of ghrelin. In some embodiment, ghrelin is thesole active agent. In some embodiments, the ghrelin is administered tothe subject in an amount that provides blood levels of ghrelin that areat least 1.0, 1.5, 2.0 or more times greater than endogenous ghrelinblood levels of the subject, thereby treating the mBI. In someembodiments, the ghrelin is administered in an amount that providesblood levels of ghrelin that are at least 2.0 times greater thanendogenous ghrelin blood levels of the subject, thereby treating themBI.

In some embodiments, the subject is free of intracranial hypertension.In some embodiments, the subject is free of blood brain barrier vascularpermeability. In some embodiments, the subject is free of necrosis ofbrain tissue. In some embodiment, the subject is free of massive braintissue or cellular damage associated with brain injury. In someembodiment, the subject is free of severe neuronal inflammation orsignificant cellular or vasogenic edema in the subject's head. In someembodiments, the subject is free of injury-associated gross anatomicalchanges to the subject's brain.

In some embodiments, the subject is free of intracranial hypertension,blood brain barrier vascular permeability, or necrosis of brain tissue.In some embodiments, the subject is free of massive brain tissue orcellular damage associated with brain injury, severe neuronalinflammation or significant cellular or vasogenic edema in the subject'shead, or injury-associated gross anatomical changes to the subject'sbrain.

In some embodiments, the subject is free of intracranial hypertension,blood brain barrier vascular permeability, necrosis of brain tissue,massive brain tissue or cellular damage associated with brain injury,severe neuronal inflammation or significant cellular or vasogenic edemain the subject's head, or injury-associated gross anatomical changes tothe subject's brain. In some embodiments, the subject is free ofintracranial hypertension, blood brain barrier vascular permeability,necrosis of brain tissue, massive brain tissue or cellular damageassociated with brain injury, severe neuronal inflammation orsignificant cellular or vasogenic edema in the subject's head, andinjury-associated gross anatomical changes to the subject's brain orcombinations thereof.

In some embodiments, mBI is associated with loss of cognitive or motorskills. In some embodiments, mBI is associated metabolic derangements.In some embodiments, mBI is associated with chronic traumaticencephalopathy (CTE). In some embodiments, mBI is associated withneuroconnectivity damage. In some embodiments, mBI is associated withencephalopathic and/or psychological changes. In some embodiments, mBIis associated with loss of consciousness.

In some embodiments, mBI is associated with loss of cognitive or motorskills and metabolic derangements. In some embodiment, mBI is associatedwith chronic traumatic encephalopathy (CTE) and neuroconnectivitydamage. In some embodiments, mBI is associated with psychologicalchanges and loss of consciousness. In some embodiments, mBI isassociated with loss of cognitive or motor skills, metabolicderangements, chronic traumatic encephalopathy (CTE), neuroconnectivitydamage, psychological changes, or loss of consciousness. In someembodiments, mBI is associated with loss of cognitive or motor skills,metabolic derangements, chronic traumatic encephalopathy (CTE),neuroconnectivity damage, psychological changes, and loss ofconsciousness or combination thereof.

The present invention provides a method for reducing, inhibiting and/orminimizing damage to neurons associated with oxidative stress andoverproduction of reactive oxidative species (ROS) with one or moreincidence of mild brain injury (mBI) or concussion in a subject,comprising administering to the subject a therapeutically effectiveamount of ghrelin, wherein the ghrelin so administered reduces oxidativestress and overproduction of ROS, thereby inhibiting damage to neuronsassociated with oxidative stress and overproduction of reactiveoxidative species (ROS) with one or more incidence of the mBI orconcussion in the subject. In one embodiment, the ghrelin has a carbon14 (C14) content of less than 1 part per trillion (ppt). In oneembodiment, the ghrelin is the sole active agent. In one embodiment, theghrelin has a carbon 14 (C14) content of less than 1 part per trillion(ppt) and ghrelin is the sole active agent.

The present invention further provides a method for inhibiting damage toneurons associated with oxidative stress and overproduction of reactiveoxidative species (ROS) with one or more incidence of mild brain injury(mBI), including concussion, in a subject, comprising administering tothe subject a therapeutically effective amount of ghrelin, wherein theghrelin so administered reduces oxidative stress and overproduction ofROS and wherein the ghrelin is the sole active agent, thereby inhibitingdamage to neurons associated with oxidative stress and overproduction ofreactive oxidative species (ROS) with one or more incidence of the mBIor concussion in the subject.

The present invention additionally provides a method for inhibitingdamage to neurons associated with oxidative stress and overproduction ofreactive oxidative species (ROS) with one or more incidence of mildbrain injury (mBI) or concussion in a subject, comprising administeringto the subject a therapeutically effective amount of ghrelin having acarbon 14 (C14) content of less than 1 part per trillion (ppt), whereinthe ghrelin so administered reduces oxidative stress and overproductionof ROS, thereby inhibiting damage to neurons associated with oxidativestress and overproduction of reactive oxidative species (ROS) with oneor more incidence of the mBI or concussion in the subject.

The present invention also provides a method for inhibiting damage toneurons associated with oxidative stress and overproduction of reactiveoxidative species (ROS) with one or more incidence of mild brain injury(mBI) or concussion in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin having a carbon 14(C14) content of less than 1 part per trillion (ppt), wherein theghrelin so administered reduces oxidative stress and overproduction ofROS and wherein the ghrelin is the sole active agent, thereby inhibitingdamage to neurons associated with oxidative stress and overproduction ofreactive oxidative species (ROS) with one or more incidence of the mBIor concussion in the subject

Additionally, the present invention discloses a method of restoringintracellular function after mild brain injury (mBI) in a subject,comprising administering to the subject a therapeutically effectiveamount of ghrelin so as to decrease ROS and improve glucose uptake ofbrain or neuronal cells and axons, thereby restoring intracellularfunction after mBI in the subject. In one embodiment, the ghrelin has acarbon 14 (C14) content of less than 1 part per trillion (ppt). In oneembodiment, the ghrelin is the sole active agent. In one embodiment, theghrelin has a carbon 14 (C14) content of less than 1 part per trillion(ppt) and is the sole active agent.

The present invention further provides a method of restoringintracellular function after mild brain injury (mBI) in a subject,comprising administering to the subject a therapeutically effectiveamount of ghrelin so as to decrease ROS and improve glucose uptake ofbrain or neuronal cells and axons, wherein the ghrelin is the soleactive agent, thereby restoring intracellular function after mBI in thesubject.

The present invention further still provides a method of restoringintracellular function after mild brain injury (mBI) in a subject,comprising administering to the subject a therapeutically effectiveamount of ghrelin having a carbon 14 (C14) content of less than 1 partper trillion (ppt) so as to decrease ROS and improve glucose uptake ofbrain or neuronal cells and axons, thereby restoring intracellularfunction after mBI in the subject.

The present invention also provides a method of restoring intracellularfunction after mild brain injury (mBI) in a subject, comprisingadministering to the subject a therapeutically effective amount ofghrelin having a carbon 14 (C14) content of less than 1 part pertrillion (ppt) so as to decrease ROS and improve glucose uptake of brainor neuronal cells and axons, wherein the ghrelin is the sole activeagent, thereby restoring intracellular function after mBI in thesubject.

Further, the present invention provides a method of treating mild braininjury (mBI) in a subject free of brain edema, intracranialhypertension, blood brain barrier vascular permeability, necrosis, ormassive tissue or cellular damage associated with brain injury,comprising administering to the subject a therapeutically effectiveamount of ghrelin, thereby treating the mBI. In one embodiment, theghrelin has a carbon 14 (C14) content of less than 1 part per trillion(ppt). In one embodiment, the ghrelin is the sole active agent. In oneembodiment, the ghrelin has a carbon 14 (C14) content of less than 1part per trillion (ppt) and is the sole active agent.

The present invention additionally provides a method of treating mildbrain injury (mBI) in a subject free of brain edema, intracranialhypertension, blood brain barrier vascular permeability, necrosis, ormassive tissue or cellular damage associated with brain injury,comprising administering to the subject a therapeutically effectiveamount of ghrelin, wherein the ghrelin is the sole active agent, therebytreating the mBI. The present invention discloses a method of treatingmild brain injury (mBI) in a subject free of brain edema, intracranialhypertension, blood brain barrier vascular permeability, necrosis, ormassive tissue or cellular damage associated with brain injury,comprising administering to the subject a therapeutically effectiveamount of ghrelin having a carbon 14 (C14) content of less than 1 partper trillion (ppt), thereby treating the mBI.

The present invention yet also provides a method of treating mild braininjury (mBI) in a subject free of brain edema, intracranialhypertension, blood brain barrier vascular permeability, necrosis, ormassive tissue or cellular damage associated with brain injury,comprising administering to the subject a therapeutically effectiveamount of ghrelin having a carbon 14 (C14) content of less than 1 partper trillion (ppt), wherein the ghrelin is the sole active agent,thereby treating the mBI.

Also, the present invention provides a method of treating mild braininjury (mBI) in a subject free of brain edema, intracranialhypertension, blood brain barrier vascular permeability, necrosis ormassive tissue and cellular damage associated with brain injury,comprising administering to the subject a therapeutically effectiveamount of ghrelin in an amount that provides blood levels of ghrelinthat are at least 1.5 times greater than endogenous ghrelin blood levelsof the subject, thereby treating the mBI. In one embodiment, the ghrelinhas a carbon 14 (C14) content of less than 1 part per trillion (ppt). Inone embodiment, the ghrelin is the sole active agent. In one embodiment,the ghrelin has a carbon 14 (C14) content of less than 1 part pertrillion (ppt) and is the sole active agent.

The present invention provides a method of treating mild brain injury(mBI) in a subject free of brain edema, intracranial hypertension, bloodbrain barrier vascular permeability, necrosis or massive tissue andcellular damage associated with brain injury, comprising administeringto the subject a therapeutically effective amount of ghrelin in anamount that provides blood levels of ghrelin that are at least 1.5 timesgreater than endogenous ghrelin blood levels of the subject, wherein theghrelin is the sole active agent, thereby treating the mBI.

The present invention yet further provides a method of treating mildbrain injury (mBI) in a subject free of brain edema, intracranialhypertension, blood brain barrier vascular permeability, necrosis ormassive tissue and cellular damage associated with brain injury,comprising administering to the subject a therapeutically effectiveamount of ghrelin having a carbon 14 (C14) content of less than 1 partper trillion (ppt) and in an amount that provides blood levels ofghrelin that are at least 1.5 times greater than endogenous ghrelinblood levels of the subject, thereby treating the mBI.

The present invention also provides a method of treating mild braininjury (mBI) in a subject free of brain edema, intracranialhypertension, blood brain barrier vascular permeability, necrosis ormassive tissue and cellular damage associated with brain injury,comprising administering to the subject a therapeutically effectiveamount of ghrelin having a carbon 14 (C14) content of less than 1 partper trillion (ppt) and in an amount that provides blood levels ofghrelin that are at least 1.5 times greater than endogenous ghrelinblood levels of the subject, wherein the ghrelin is the sole activeagent, thereby treating the mBI.

The present invention further provides a method of treating metabolicderangements associated with mild brain injury (mBI) or concussion in asubject, comprising administering to the subject a therapeuticallyeffective amount of ghrelin, thereby treating metabolic derangementsassociated with mBI or concussion in the subject. In one embodiment, theghrelin has a carbon 14 (C14) content of less than 1 part per trillion(ppt). In one embodiment, the ghrelin is the sole active agent. In oneembodiment, the ghrelin has a carbon 14 (C14) content of less than 1part per trillion (ppt) and is the sole active agent.

The present invention discloses a method of treating metabolicderangements associated with mild brain injury (mBI) or concussion in asubject, comprising administering to the subject a therapeuticallyeffective amount of ghrelin, wherein the ghrelin is the sole activeagent, thereby treating metabolic derangements associated with mBI orconcussion in the subject.

The present invention provides a method of treating metabolicderangements associated with mild brain injury (mBI) or concussion in asubject, comprising administering to the subject a therapeuticallyeffective amount of ghrelin having a carbon 14 (C14) content of lessthan 1 part per trillion (ppt), thereby treating metabolic derangementsassociated with mBI or concussion in the subject.

The present invention further still provides a method of treatingmetabolic derangements associated with mild brain injury (mBI) orconcussion in a subject, comprising administering to the subject atherapeutically effective amount of ghrelin having a carbon 14 (C14)content of less than 1 part per trillion (ppt), wherein the ghrelin isthe sole active agent, thereby treating metabolic derangementsassociated with mBI or concussion in the subject.

The present invention provides a method of treating metabolicderangements associated with mild brain injury (mBI) or concussion in asubject, comprising administering to the subject a therapeuticallyeffective amount of ghrelin in an amount that provides blood levels ofghrelin that are at least 1.5 times greater than endogenous ghrelinblood levels of the subject, thereby treating metabolic derangementsassociated with mBI or concussion in the subject. In one embodiment, theghrelin has a carbon 14 (C14) content of less than 1 part per trillion(ppt). In one embodiment, the ghrelin is the sole active agent. In oneembodiment, the ghrelin has a carbon 14 (C14) content of less than 1part per trillion (ppt) and is the sole active agent.

The present invention provides a method of treating metabolicderangements associated with mild brain injury (mBI) or concussion in asubject, comprising administering to the subject a therapeuticallyeffective amount of ghrelin in an amount that provides blood levels ofghrelin that are at least 1.5 times greater than endogenous ghrelinblood levels of the subject, wherein the ghrelin is the sole activeagent, thereby treating metabolic derangements associated with mBI orconcussion in the subject.

The present invention provides a method of treating metabolicderangements associated with mild brain injury (mBI) or concussion in asubject, comprising administering to the subject a therapeuticallyeffective amount of ghrelin in an amount that provides blood levels ofghrelin that are at least 1.5 times greater than endogenous ghrelinblood levels of the subject, wherein the ghrelin has a carbon 14 (C14)content of less than 1 part per trillion (ppt), thereby treatingmetabolic derangements associated with mBI or concussion in the subject.

The present invention provides a method of treating metabolicderangements associated with mild brain injury (mBI) or concussion in asubject, comprising administering to the subject a therapeuticallyeffective amount of ghrelin in an amount that provides blood levels ofghrelin that are at least 1.5 times greater than endogenous ghrelinblood levels of the subject, wherein the ghrelin has a carbon 14 (C14)content of less than 1 part per trillion (ppt) and is the sole activeagent, thereby treating metabolic derangements associated with mBI orconcussion in the subject.

The present invention provides a method for preventing damage to neuronsassociated with oxidative stress and overproduction of reactiveoxidative species (ROS) with one or more incidence of mild brain injury(mBI) or concussion in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin, wherein ghrelintreatment reduces oxidative stress and overproduction of ROS, therebypreventing damage to neurons associated with oxidative stress andoverproduction of reactive oxidative species (ROS) with one or moreincidence of the mBI or concussion in the subject. In one embodiment,the ghrelin has a carbon 14 (C14) content of less than 1 part pertrillion (ppt). In one embodiment, the ghrelin is the sole active agent.In one embodiment, the ghrelin has a carbon 14 (C14) content of lessthan 1 part per trillion (ppt) and is the sole active agent. The presentinvention discloses a method for preventing damage to neurons associatedwith oxidative stress and overproduction of reactive oxidative species(ROS) with one or more incidence of mild brain injury (mBI) orconcussion in a subject, comprising administering to the subject atherapeutically effective amount of ghrelin, wherein ghrelin treatmentreduces oxidative stress and overproduction of ROS and wherein theghrelin is the sole active agent, thereby preventing damage to neuronsassociated with oxidative stress and overproduction of reactiveoxidative species (ROS) with one or more incidence of the mBI orconcussion in the subject.

The present invention provides a method for preventing damage to neuronsassociated with oxidative stress and overproduction of reactiveoxidative species (ROS) with one or more incidence of mild brain injury(mBI) or concussion in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin having a carbon 14(C14) content of less than 1 part per trillion (ppt), wherein ghrelintreatment reduces oxidative stress and overproduction of ROS, therebypreventing damage to neurons associated with oxidative stress andoverproduction of reactive oxidative species (ROS) with one or moreincidence of the mBI or concussion in the subject.

The present invention provides a method for preventing damage to neuronsassociated with oxidative stress and overproduction of reactiveoxidative species (ROS) with one or more incidence of mild brain injury(mBI) or concussion in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin having a carbon 14(C14) content of less than 1 part per trillion (ppt), wherein ghrelintreatment reduces oxidative stress and overproduction of ROS and whereinthe ghrelin is the sole active agent, thereby preventing damage toneurons associated with oxidative stress and overproduction of reactiveoxidative species (ROS) with one or more incidence of the mBI orconcussion in the subject.

The present invention provides a method for preventing damage to neuronsassociated with oxidative stress and overproduction of reactiveoxidative species (ROS) with one or more incidence of mild brain injury(mBI) or concussion in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin in an amount thatprovides blood levels of ghrelin that are at least 1.5 times greaterthan endogenous ghrelin blood levels of the subject, wherein ghrelintreatment reduces oxidative stress and overproduction of ROS, therebypreventing damage to neurons associated with oxidative stress andoverproduction of reactive oxidative species (ROS) with one or moreincidence of the mBI or concussion in the subject. In one embodiment,the ghrelin has a carbon 14 (C14) content of less than 1 part pertrillion (ppt). In one embodiment, the ghrelin is the sole active agent.In one embodiment, the ghrelin has a carbon 14 (C14) content of lessthan 1 part per trillion (ppt) and is the sole active agent.

The present invention provides a method for preventing damage to neuronsassociated with oxidative stress and overproduction of reactiveoxidative species (ROS) with one or more incidence of mild brain injury(mBI) or concussion in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin in an amount thatprovides blood levels of ghrelin that are at least 1.5 times greaterthan endogenous ghrelin blood levels of the subject, wherein ghrelintreatment reduces oxidative stress and overproduction of ROS and whereinthe ghrelin is the sole active agent, thereby preventing damage toneurons associated with oxidative stress and overproduction of reactiveoxidative species (ROS) with one or more incidence of the mBI orconcussion in the subject.

The present invention provides a method for preventing damage to neuronsassociated with oxidative stress and overproduction of reactiveoxidative species (ROS) with one or more incidence of mild brain injury(mBI) or concussion in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin in an amount thatprovides blood levels of ghrelin that are at least 1.5 times greaterthan endogenous ghrelin blood levels of the subject, wherein ghrelintreatment reduces oxidative stress and overproduction of ROS and whereinthe ghrelin has a carbon 14 (C14) content of less than 1 part pertrillion (ppt), thereby preventing damage to neurons associated withoxidative stress and overproduction of reactive oxidative species (ROS)with one or more incidence of the mBI or concussion in the subject.

The present invention provides a method for reducing damage to neuronsassociated with oxidative stress and overproduction of reactiveoxidative species (ROS) with one or more incidence of mild brain injury(mBI) or concussion in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin in an amount thatprovides blood levels of ghrelin that are at least 1.5 times greaterthan endogenous ghrelin blood levels of the subject, wherein ghrelintreatment reduces oxidative stress and overproduction of ROS and whereinthe ghrelin has a carbon 14 (C14) content of less than 1 part pertrillion (ppt) and is the sole active agent, thereby preventing damageto neurons associated with oxidative stress and overproduction ofreactive oxidative species (ROS) with one or more incidence of the mBIor concussion in the subject.

The present invention provides a method of treating mild brain injury(mBI) in a subject, free of acute neuronal histological changes, severeneuronal inflammation or significant cellular or vasogenic edema,comprising administering to the subject a therapeutically effectiveamount of ghrelin, thereby treating the mBI in the subject free of acuteneuronal histological changes, severe neuronal inflammation orsignificant cellular or vasogenic edema. In one embodiment, the ghrelinhas a carbon 14 (C14) content of less than 1 part per trillion (ppt). Inone embodiment, the ghrelin is the sole active agent. In one embodiment,the ghrelin has a carbon 14 (C14) content of less than 1 part pertrillion (ppt) and is the sole active agent.

The present invention provides a method of treating mild brain injury(mBI) in a subject, free of acute neuronal histological changes, severeneuronal inflammation or significant cellular or vasogenic edema,comprising administering to the subject a therapeutically effectiveamount of ghrelin, wherein the ghrelin is the sole active agent, therebytreating the mBI in the subject free of acute neuronal histologicalchanges, severe neuronal inflammation or significant cellular orvasogenic edema.

The present invention provides a method of treating mild brain injury(mBI) in a subject, free of acute neuronal histological changes, severeneuronal inflammation or significant cellular or vasogenic edema,comprising administering to the subject a therapeutically effectiveamount of ghrelin having a carbon 14 (C14) content of less than 1 partper trillion (ppt), thereby treating the mBI in the subject free ofacute neuronal histological changes, severe neuronal inflammation orsignificant cellular or vasogenic edema.

The present invention provides a method of treating mild brain injury(mBI) in a subject, free of acute neuronal histological changes, severeneuronal inflammation or significant cellular or vasogenic edema,comprising administering to the subject a therapeutically effectiveamount of ghrelin having a carbon 14 (C14) content of less than 1 partper trillion (ppt), wherein the ghrelin is the sole active agent,thereby treating the mBI in the subject free of acute neuronalhistological changes, severe neuronal inflammation or significantcellular or vasogenic edema.

The present invention provides a method of treating mild brain injury(mBI) in a subject, comprising administering to the subject, free ofacute neuronal histological changes, severe neuronal inflammation orsignificant cellular or vasogenic edema, a therapeutically effectiveamount of ghrelin in an amount that provides blood levels of ghrelinthat are at least 1.5 times greater than endogenous ghrelin blood levelsof the subject, thereby treating the mBI in the subject free of acuteneuronal histological changes, severe neuronal inflammation orsignificant cellular or vasogenic edema. In one embodiment, the ghrelinhas a carbon 14 (C14) content of less than 1 part per trillion (ppt). Inone embodiment, the ghrelin is the sole active agent. In one embodiment,the ghrelin has a carbon 14 (C14) content of less than 1 part pertrillion (ppt) and is the sole active agent.

The present invention provides a method of treating mild brain injury(mBI) in a subject, comprising administering to the subject, free ofacute neuronal histological changes, severe neuronal inflammation orsignificant cellular or vasogenic edema, a therapeutically effectiveamount of ghrelin in an amount that provides blood levels of ghrelinthat are at least 1.5 times greater than endogenous ghrelin blood levelsof the subject, wherein the ghrelin is the sole active agent, therebytreating the mBI in the subject free of acute neuronal histologicalchanges, severe neuronal inflammation or significant cellular orvasogenic edema.

The present invention provides a method of treating mild brain injury(mBI) in a subject, comprising administering to the subject, free ofacute neuronal histological changes, severe neuronal inflammation orsignificant cellular or vasogenic edema, a therapeutically effectiveamount of ghrelin in an amount that provides blood levels of ghrelinthat are at least 1.5 times greater than endogenous ghrelin blood levelsof the subject, wherein the ghrelin has a carbon 14 (C14) content ofless than 1 part per trillion (ppt), thereby treating the mBI in thesubject free of acute neuronal histological changes, severe neuronalinflammation or significant cellular or vasogenic edema.

The present invention provides a method of treating mild brain injury(mBI) in a subject, comprising administering to the subject, free ofacute neuronal histological changes, severe neuronal inflammation orsignificant cellular or vasogenic edema, a therapeutically effectiveamount of ghrelin in an amount that provides blood levels of ghrelinthat are at least 1.5 times greater than endogenous ghrelin blood levelsof the subject, wherein the ghrelin the ghrelin has a carbon 14 (C14)content of less than 1 part per trillion (ppt) and is the sole activeagent, thereby treating the mBI in the subject free of acute neuronalhistological changes, severe neuronal inflammation or significantcellular or vasogenic edema.

The present invention provides a method of treating or reducing symptomsof mild brain injury (mBI) in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin, thereby treatingor reducing symptoms of the mBI. In one embodiment, the ghrelin has acarbon 14 (C14) content of less than 1 part per trillion (ppt). In oneembodiment, the ghrelin is the sole active agent. In one embodiment, theghrelin has a carbon 14 (C14) content of less than 1 part per trillion(ppt) and is the sole active agent.

The present invention provides a method of treating or reducing symptomsof mild brain injury (mBI) in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin, wherein theghrelin is the sole active agent, thereby treating or reducing symptomsof the mBI.

The present invention provides a method of treating or reducing symptomsof mild brain injury (mBI) in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin having a carbon 14(C14) content of less than 1 part per trillion (ppt), thereby treatingor reducing symptoms of the mBI.

The present invention provides a method of treating or reducing symptomsof mild brain injury (mBI) in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin having a carbon 14(C14) content of less than 1 part per trillion (ppt), wherein theghrelin is the sole active agent, thereby treating or reducing symptomsof the mBI.

The present invention provides a method of treating or reducing symptomsof mild brain injury (mBI) in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin in an amount thatprovides blood levels of ghrelin that are at least 1.5 times greaterthan endogenous ghrelin blood levels of the subject, thereby treating orreducing symptoms of the mBI. In one embodiment, the ghrelin has acarbon 14 (C14) content of less than 1 part per trillion (ppt). In oneembodiment, the ghrelin is the sole active agent. In one embodiment, theghrelin has a carbon 14 (C14) content of less than 1 part per trillion(ppt) and is the sole active agent.

The present invention provides a method of treating or reducing symptomsof mild brain injury (mBI) in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin in an amount thatprovides blood levels of ghrelin that are at least 1.5 times greaterthan endogenous ghrelin blood levels of the subject, wherein the ghrelinis the sole active agent, thereby treating or reducing symptoms of themBI.

The present invention provides a method of treating or reducing symptomsof mild brain injury (mBI) in a subject, comprising administering to thesubject a therapeutically effective amount of ghrelin in an amount thatprovides blood levels of ghrelin that are at least 1.5 times greaterthan endogenous ghrelin blood levels of the subject, wherein the ghrelinhas a carbon 14 (C14) content of less than 1 part per trillion (ppt),thereby treating or reducing symptoms of the mBI. The present inventionprovides a method of treating or reducing symptoms of mild brain injury(mBI) in a subject, comprising administering to the subject atherapeutically effective amount of ghrelin in an amount that providesblood levels of ghrelin that are at least 1.5 times greater thanendogenous ghrelin blood levels of the subject, wherein the ghrelin hasa carbon 14 (C14) content of less than 1 part per trillion (ppt) and isthe sole active agent, thereby treating or reducing symptoms of the mBI.

In some embodiments, the symptoms of mBI are any of headache, “pressurein head,” neck pain, nausea or vomiting, dizziness, blurred vision,balance problems, sensitivity to light, sensitivity to noise, feelingslowed down, feeling like “in a fog,” “don't feel right,” difficultyconcentrating, difficulty remembering, fatigue or low energy, confusion,drowsiness, loss of consciousness, trouble falling asleep, moreemotional, irritability, sadness and being nervous or anxious.

As discussed herein, ghrelin can be used to treat and/or reduce theseverity of mild brain injuries, such as for example, concussions. Theghrelin can be provided or administered as described herein. Asdescribed elsewhere herein, ghrelin can be the sole active agentadministered to a subject. For example, ghrelin can be the only activeingredient for treating the mBI or reducing the severity of the mBI. Insome embodiments, additional active agents are specifically excluded.For example, in some of the methods herein where ghrelin isadministered, the methods can specifically exclude the use oradministration of other compounds, such as, for example, selectiveandrogen receptor modulators, steroid receptor modulators, ligand for aglucocorticoid receptor, or a modulator of the NF-kB signaling pathway.

In the embodiments described herein it should be understood that mBI isdifferent from and is not severe TBI.

In some embodiments, mBI or concussion is diagnosed by non-invasivefunctional imaging following impact. In some embodiments, mBI orconcussion is diagnosed by neuropsychological assessment followingimpact. In some embodiment, mBI or concussion is diagnosed byneuroanatomical assessment of the brain following impact. In someembodiment, the neuroanatomical assessment of the brain following impactis at the cellular level or molecular level. In some embodiment, mBI orconcussion is diagnosed by cognitive assessment following impact. Insome embodiment, mBI or concussion is diagnosed by assessment ofalertness following impact. In some embodiments, mBI and concussion canbe diagnosed and/or assessed by evaluating one or more of consciousness,assessment of alertness, assessment of orientation, assessment ofcognitive function, assessment of psychological state,neuropsychological assessment.

In some embodiments, mBI or concussion is diagnosed using a Glasgow ComaScale (GCS) having a score of 13 to 15. In some embodiments, mBI orconcussion is diagnosed using a Glasgow Coma Scale (GCS) having a scoreof 13 or greater.

In contrast, by way of example, severe TBI generally has a GCS score of8 or below.

In some embodiments, an intravenous injection of ghrelin is employed.The administration route must ensure that the non-degraded, bioactiveform of the peptide will be the dominating form in the circulation,which will reach and stimulate the ghrelin receptors in order to obtainthe maximum effect of ghrelin treatment on mBI. In some embodiments,ghrelin is administered within about 30 minutes of the incident thatresults in mBI. In some embodiments, ghrelin is administered withinabout 30 minutes to about 2 hours of the incident that results in mBI.In some embodiments, ghrelin is administered within about 30 minutes toabout 6 hours of the incident that results in mBI. In some embodiments,ghrelin is administered within about 30 minutes to about 12 hours of theincident that results in mBI.

A typical dosage is in a concentration equivalent to from 10 ng to 10 mgghrelin per kg bodyweight. In some embodiments, ghrelin is administeredin a concentration equivalent to from about 0.1 μg to about 1 mg ghrelinper kg bodyweight, such as from about 0.5 μg to about 0.5 mg ghrelin perkg bodyweight, such as from about 1.0 μg to about 0.1 mg ghrelin per kgbodyweight, such as from about 1.0 μg to about 50 μg ghrelin per kgbodyweight, such as from about 1.0 μg to about 10 μg ghrelin per kgbodyweight. In some embodiments, about 10 μg ghrelin powder isreconstituted in about 100 μL of a sterile saline solution beforeadministration. In some embodiments, the sterile saline solution iscontained in an IV bag for ease of delivery.

In some embodiments, the ghrelin and ghrelin or ghrelin agonist is usedin an assay to assess the ability of candidate compounds to effectincreased uncoupling protein-2 (UCP-2) expression including increasedUCP-2 expression in mitochondria. In such assays, ghrelin is used as acontrol to determine the relative efficacy of the candidate compound orcompounds. Suitable assays include by way of example only competitiveassays for binding of a candidate compound or compounds to growthhormone secretagogue receptor 1a (i.e., GHSR-1a) in the presence ofghrelin as well as frontal affinity chromatography.

Any competitive binding assay known in the art is applicable for bindingof a candidate compound or compounds to growth hormone secretagoguereceptor in the presence of ghrelin, using either heterogeneous orhomogeneous methods, with one or more reagents, and with labels anddetection methods. By way of non-limiting example, detection methods mayinclude radioactive methods; enzyme techniques using intact enzymes ofmany types including, for example, β-galactosidase, glucose 6-phosphatedehydrogenase, alkaline phosphatase, horseradish peroxidase, or glucoseoxidase; techniques using enzyme fragments, such as β-galactosidasecomplementation assays; detection systems including chromogenicsubstrates; fluorescent methods detected by direct fluorescence,time-resolved fluorescence, fluorescence polarization, or fluorescenceenergy transfer; and chemical or bioluminescence detection systems.

In some embodiments, frontal affinity chromatography (FAC) can be usedfor screening of compound libraries. The basic premise of FAC is thatcontinuous infusion of a compound will allow for equilibration of theligand between the free and bound states, where the preciseconcentration of free ligand is known. The detection of compoundseluting from the column can be accomplished using methods such asfluorescence, radioactivity, or electrospray mass spectrometry. Theformer two methods usually make use of either a labeled library, or usea labeled indicator compound, which competes against known unlabeledcompounds, getting displaced earlier if a stronger binding ligand ispresent.

In some embodiments, a patient suffering loss of cognitive or motorskills due to mBI and, in particular, repetitive mBI, can be monitoredfor therapy or progression of such skills by correlating the ghrelinlevel in the patient's brain over time. As the ghrelin levels decrease,there will be an increased need for intervention.

This invention also provides for methods for measuring ghrelin levelsbefore starting a sport or activity, for example prior to the beginningof football season (or any other sport or activity, including thoselisted elsewhere herein), and monitoring ghrelin levels during theseason to ascertain if the player or participant is at a level notqualified to play or participate. The methods can include the use of anysuitable measurement or assay technique for measuring ghrelin levels,such as from blood to determine if blood levels correlate to brainlevels.

With the benefit of the instant embodiments, the skilled artisan canselect any suitable technique for measuring ghrelin levels. A number ofassays known in the art for measuring a protein or hormone level areapplicable for measuring ghrelin levels. By way of non-limiting example,assays such as a blood sugar test by extracting a drop of blood andputting it into a device, can quantitatively assess the amount ofghrelin, or an assay involving measuring a range of substances whereby aspecific reaction chemistry is followed photo-metrically with time, forexample by utilizing an antibody specific to ghrelin that is coated ontolatex particles and measuring the increased turbidity that is producedwhen ghrelin being measured promotes aggregation of the latex particlesas the reaction between ghrelin and anti-ghrelin antibody proceeds. Thismeasurement of increasing turbidity can be achieved using a conventionalphotometer and using the associated scientific principles of photometricmeasurements. Such concentration dependent turbidity is then compared tothat produced by standards which are established in the art.

Further compatible, but non-limiting, methodologies include carrying outa series of enzyme-linked reactions in solution, where ghrelin in theplasma fraction of a whole blood sample is altered by an enzyme-promotedreaction to ultimately derive a colored dye from colorless reactionconstituents. The color is developed in a time dependent way andmonitored photo-metrically. This measurement of color change can also beachieved using a conventional photometer using the associated scientificprinciples of photometric measurements. Such concentration dependentchange in transmission is then compared to that produced by standards.

In whole blood samples, hematocrit or percentage of red blood cells byvolume in the whole blood sample is a variable and can be taken intoaccount when analyzing ghrelin levels that are present in the plasmacomponent. As the hematocrit of a patient's blood rises, so does thevolume of plasma in a fixed volume sample, which is introduced into thetest device decreases and vice versa. Since it is the plasma componentwhich exclusively carries the ghrelin levels being measured, then thelower the volume of plasma component added to the reaction mix, thelower the resulting concentration of the substance being measured inthat reaction mix and the resulting assayed value and vice versa.

Any analysis that produces a concentration of a plasma substance inwhole blood may be corrected for variations in hematocrit to give a trueplasma concentration. It can be most useful in these situations tomeasure two substances, one of which is ghrelin under investigation andthe other which is considered to be a marker by which to estimate ornormalize the sample hematocrit. The hemoglobin concentration of wholeblood, after red blood cells are lysed, is directly proportional to thered blood cell volume in the whole blood sample.

Pharmaceutical Compositions

Ghrelin described herein can be formulated as a pharmaceuticalcomposition, e.g., flash frozen or lyophilized for storage and/ortransport. In some embodiments, the compound can be in a compositionwith sterile saline, for example. In some embodiments, a ghrelin can bereconstituted in such saline or other acceptable diluent. In someembodiments, about 10 μg ghrelin powder is reconstituted in about 100 pisaline solution before administration. In addition, the composition canbe administered alone or in combination with a carrier, such as apharmaceutically acceptable carrier or a biocompatible scaffold.Compositions of the invention may be conventionally administeredparenterally, by injection, for example, intravenously, subcutaneously,or intramuscularly. Additional formulations which are suitable for othermodes of administration include oral formulations. Oral formulationsinclude such normally employed excipients such as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain about10% to about 95% of active ingredient, about 25% to about 70%.

Typically, compositions are administered in a manner compatible with thedosage formulation, and in such amount as will be therapeuticallyeffective for the disease or condition by treated. The quantity to beadministered depends on the subject to be treated. Precise amounts ofthe composition to be administered depend on the judgment of thepractitioner. Suitable regimes for initial administration and boostersare also variable, but are typified by an initial administrationfollowed by subsequent administrations.

In some embodiments, additional pharmaceutical compositions areadministered to a subject to support or augment the compositions asdescribed herein. Different aspects of the present invention involveadministering an effective amount of the composition to a subject.Additionally, such compositions can be administered in combination withother agents. Such compositions will generally be dissolved or dispersedin a pharmaceutically acceptable carrier or aqueous medium.

The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic, or other untoward reaction whenadministered to an animal, or human. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredients, its use in immunogenic and therapeutic compositionsis contemplated.

Suitable pharmaceutical carriers include inert solid diluents orfillers, sterile aqueous solution and various organic solvents. Examplesof solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc,gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or loweralkyl ethers of cellulose. Examples of liquid carriers are syrup, peanutoil, olive oil, phospholipids, fatty acids, fatty acid amines,polyoxyethylene or water. Nasal aerosol or inhalation formulations maybe prepared, for example, as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, employing fluorocarbons, and/or employing othersolubilizing or dispersing agents.

An effective amount of therapeutic composition is determined based onthe intended goal. The term “unit dose” or “dosage” refers to physicallydiscrete units suitable for use in a subject, each unit containing apredetermined quantity of the composition calculated to produce thedesired responses discussed above in association with itsadministration, i.e., the appropriate route and regimen. The quantity tobe administered, both according to number of treatments and unit dose,depends on the result and/or protection desired. Precise amounts of thecomposition also depend on the judgment of the practitioner and arepeculiar to each individual. Factors affecting dose include physical andclinical state of the subject, route of administration, intended goal oftreatment (alleviation of symptoms versus cure), and potency, stability,and toxicity of the particular composition. Upon formulation, solutionswill be administered in a manner compatible with the dosage formulationand in such amount as is therapeutically or prophylactically effective.The formulations are easily administered in a variety of dosage forms,such as the type of injectable solutions described above.

Ghrelin compositions can be produced using techniques well known in theart. For example, a polypeptide region of ghrelin can be chemically orbiochemical synthesized and modified. Techniques for chemical synthesisof polypeptides are well known in the art (Lee V. H. L. in “Peptide andProtein Drug Delivery”, New York, N.Y., M. Dekker, 1990). Examples oftechniques for biochemical synthesis involving the introduction of anucleic acid into a cell and expression of nucleic acids are provided inAusubel F. M. et al., “Current Protocols in Molecular Biology”, JohnWiley, 1987-1998, and Sambrook J. et al., “Molecular Cloning, ALaboratory Manual”, 2d Edition, Cold Spring Harbor Laboratory Press,1989, each of which is incorporated herein by reference. Anotherexemplary technique described in U.S. Pat. No. 5,304,489, incorporatedherein by reference, is the use of a transgenic mammal having mammarygland-targeted mutations, which result in the production and secretionof synthesized ghrelin in the milk of the transgenic mammal.

Ghrelin can also be produced recombinantly using routine expressionmethods known in the art. The polynucleotide encoding the desiredghrelin (e.g., primarily sequence of ghrelin as given in SEQ ID NO. 1)is operably linked to a promoter into an expression vector suitable forany convenient host. Both eukaryotic and prokaryotic host systems areused in forming recombinant ghrelin. Ghrelin is then isolated from lysedcells or from the culture medium and purified to the extent needed forits intended use. Isolated ghrelin may be modified or further modifiedat serine amino acid position 2 and/or serine amino acid position 3 byfatty acid acylation in vivo or in vitro, with the latter in vitroacylation reaction condition comprising fatty acid thioester, ghrelin,and microsomes comprising ghrelin O-acyl transferase (GOAT). In someembodiments, acyl ghrelin modified with fatty acid at serine amino acidposition 2 and/or serine amino acid position 3 is isolated from cellularor reaction components.

Ghrelin compositions can include pharmaceutically acceptable salts ofthe compounds therein. These salts will be ones which are acceptable intheir application to a pharmaceutical use, meaning that the salt willretain the biological activity of the parent compound and the salt willnot have untoward or deleterious effects in its application and use intreating diseases. Pharmaceutically acceptable salts are prepared in astandard manner.

Ghrelin compositions may be administered in the form of an alkali metalor earth alkali metal salt thereof, concurrently, simultaneously, ortogether with a pharmaceutically acceptable carrier or diluent,especially and in the form of a pharmaceutical composition thereof,whether by various routes (e.g., oral, rectal, parenteral, subcutaneous)in an effective amount.

Administration of Compositions

The present disclosure provides for a method of reducing the incidenceor severity of mBI in a subject, comprising administering to the subjectan effective amount of a compound comprising ghrelin, thereby reducingthe incidence or severity of the mBI. The present disclosure alsoprovides for methods of reducing the incidence or severity of mBI in asubject, comprising administering to the subject an effective amount ofghrelin, thereby reducing the incidence or severity of the mBI. Thisinvention further provides for methods of reducing the amount of timeneeded to recover from a mild brain injury, comprising administering toa patient suffering from a mild brain injury a therapeutically effectiveamount of ghrelin within a certain period (e.g., 72 hours) of the mildbrain injury.

In some embodiments, ghrelin is administered prior to an event oractivity with a potential for occurrence of mBI. In some embodiments,the event or activity is participation in a sporting event, physicaltraining, or combat. In some embodiments, the event or activity isbaseball, basketball, rugby, football, hockey, lacrosse, soccer,cycling, boxing, gymnastics, a martial art, a mixed martial art, amilitary exercise, automobile racing, snow skiing, snowboarding, iceskating, skateboarding, motocross, mountain biking, motorcycle and ATVriding, and the like. In some embodiments, the subject has not suffereda mBI. In some embodiments, the subject has a history of mBI or issusceptible to mBI.

In some embodiments, an administration route for a ghrelin is selectedfrom: buccal delivery, sublingual delivery, transdermal delivery,inhalation and needle-free injection, such as using the methodsdeveloped by PowderJet. For inhalation, ghrelin can be formulated usingmethods known to those skilled in the art, for example an aerosol, drypowder or solubilized such as in microdroplets, in a device intended forsuch delivery (such as commercially available devices and formulationtechnologies from Aradigm Corp. (Hayward, Calif.), Alkermes, Inc.(Cambridge, Mass.), Nektar Therapeutics (San Carlos, Calif.), WindgapMedical, Inc.(e.g., Abiliject™) or MannKind Corporation (Valencia,Calif.; e.g., Technosphere®, Dreamboat®, and Cricket™ technologies)).

In some embodiments, the ghrelin is administered via a powder or stableformulation, wherein ghrelin is formulated in a dosage form selectedfrom the group consisting of: liquid, beverage, medicated sports drink,powder, capsule, chewable tablet, caplet, swallowable tablet, buccaltablet, troche, lozenge, soft chew, solution, suspension, spray,suppository, tincture, decoction, infusion, and a combination thereof.

In some embodiments, ghrelin is administered via inhalation, oral,intravenous, parenteral, buccal, subcutaneous, transdermal, patch,sublingual, intramuscular, or intranasal. In some embodiments, ghrelinis administered in a single dose. In some embodiments, ghrelin isadministered in multi-doses. In some embodiments, ghrelin isadministered at a dosage from 10 ng/kg per day to 10 mg/kg per day (orany sub value or sub range there between, e.g., 0.1 μg/kg per day to 5mg/kg per day). In some embodiments, a dosing regimen (2 μg/kg, forexample delivered intravenously) is administered within 8 hoursfollowing injury. The dosing is a one-time dose with possible recurrentdosing based on patient symptoms.

Nasal delivery is a non-invasive route for therapeutics targeting thecentral nervous system because of relatively high permeability of nasalepithelium membrane, avoidance of hepatic first pass elimination. Nasaldelivery is easy to administer and allows for self-medication by anindividual. Nasal mucociliary clearance is an important limiting factorto nasal drug delivery. Nasal mucociliary clearance severely limits thetime allowed for drug absorption to occur and may effectively preventsustained drug administration. However, it has been documented thatnasal administration of certain hormones has resulted in a more completeadministration. In some embodiments, the present disclosure utilizesnasal delivery of ghrelin.

In some embodiments, a composition comprising ghrelin that is suitablefor nasal administration may include one or more bioadhesive polymers.Some polymers such as carbopol, can adhere onto the nasal mucosa forreasonably prolonged periods, preventing rapid nasal clearance. In someembodiments, a composition suitable for nasal administration, thepercentage of bioadhesive polymer in a suitable solution of ghrelin isabout 0.1%. In some embodiments, a composition suitable for nasaladministration, the percentage of bioadhesive polymer in a suitablesolution of ghrelin is about 0.5%. In some embodiments, a compositionsuitable for nasal administration, the percentage of bioadhesive polymerin a suitable solution of ghrelin is about 1%. In some embodiments, acomposition suitable for nasal administration, the percentage ofbioadhesive polymer in a suitable solution of ghrelin is about 5%.

In some embodiments, a composition comprising ghrelin that is suitablefor nasal administration may include one or more surfactants.Surfactants that may be used in the compositions of the presentinvention include different polyethylene glycols (PEGS) and polyethyleneglycol-derivatives. In some embodiments, a composition suitable fornasal administration, the percentage of surfactant in a suitablesolution of ghrelin is about 1%. In some embodiments, a compositionsuitable for nasal administration, the percentage of surfactant in asuitable solution of ghrelin is about 2%. In some embodiments, acomposition suitable for nasal administration, the percentage ofsurfactant in a suitable solution of ghrelin is about 5%. In someembodiments, a composition suitable for nasal administration, thepercentage of surfactant in a suitable solution of ghrelin is about 10%.

In some embodiments, a composition comprising ghrelin that is suitablefor nasal administration may include one or more buffering agents forcontrolling the pH of the composition. Buffering agents that may be usedin the compositions of the present invention include citric acid andsodium citrate dihydrate. In some embodiments, a composition suitablefor nasal administration, the percentage of buffering agent in asuitable solution of ghrelin is about 0.001%. In some embodiments, acomposition suitable for nasal administration, the percentage ofbuffering agent in a suitable solution of ghrelin is about 0.005%. Insome embodiments, a composition suitable for nasal administration, thepercentage of buffering agent in a suitable solution of ghrelin is about0.01%. In some embodiments, a composition suitable for nasaladministration, the percentage of buffering agent in a suitable solutionof ghrelin is about 0.1%.

In some embodiments, the osmolarity of the composition comprisingghrelin may be controlled by propylene glycol. When a compositioncomprising ghrelin is a gel, the composition may include a gelling agentsuch as hydroxylpropyl cellulose, carbopols, carboxymethylcellulose, andethylcellulose. In some embodiments, the composition comprising ghrelinmay include a preservative such as ethylenediaminetetraacetic acid(EDTA) and benzalkonium chloride. Non-limiting examples of suitablesolvents for compositions of the present invention include water,vegetable oil and ethanol. In some embodiments, the use of a nasalinhalant reduces the concentration required to treat mBI and preventunwanted side effects.

In some embodiments, nasal administration is a more practical means ofdelivery in a military or sport setting. In some embodiments, thepresent invention provides a method for improving the standard of carefor preventing or treating mBI in military personnel or athletes througha prophylactic and post-acute intranasal therapeutic. In someembodiments, the active ingredient of the therapeutic is ghrelin. Insome embodiments, ghrelin may be part of a formulation that is deliveredintranasally to facilitate ease of access and use in the field and tominimize the dose required further limiting side effects. Using ghrelinas a therapeutic may reduce poor outcomes following injury, especiallyneuropsychological and neurodegenerative disorders including ChronicTraumatic Encephalopathy (CTE) and Post-Traumatic Stress Disorder (PTSD)linked to repetitive brain injuries, an increasing concern for today'smilitary personnel and athletes.

In some embodiments, the present invention provides compositionscomprising ghrelin that are applied as nasal drops, eye drops and nasalsprays. For the nasal application, a solution or suspension may be usedwhich is applied as spray, i.e., in the form of a fine dispersion in airor by means of a conventional spray-squeeze bottle or pump. Suitablenontoxic pharmaceutically acceptable carriers for use in a drug deliverysystem for intranasal administration of ghrelin may include, but notlimited to, carriers used for nasal pharmaceutical formulations forother steroids, such as estrogen.

In some embodiments, formulations of the present invention may contain apreservative and/or stabilizer. These include, but not limited to,ethylene diamine tetraacetic acid (EDTA) and its alkali salts (forexample dialkali salts such as disodium salt, calcium salt,calcium-sodium salt), lower alkyl p-hydroxybenzoates, chlorhexidine (forexample in the form of the acetate or gluconate) and phenyl mercuryborate. Other suitable preservatives are: pharmaceutically usefulquaternary ammonium compounds, for example cetylpyridinium chloride,tetradecyltrimethyl ammonium bromide, generally known as “cetrimide”,N-Benzyl-N,N-dimethyl-2-{2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy}ethanaminiumchloride, generally known as “benzethonium chloride” and myristylpicolinium chloride. Each of these compounds may be used in aconcentration of about 0.002 to 0.05%, for example about 0.02%(weight/volume in liquid formulations, otherwise weight/weight). In someembodiments, preservatives among the quaternary ammonium compounds are,but not limited to, alkylbenzyl dimethyl ammonium chloride and mixturesthereof, for example, benzalkonium chloride.

In some embodiments, the present invention provides for a treatmentstrategy for athletes who have suffered a mBI that may not only reducethe time required for safe return to play but also provide protectionfrom future mBI.

Intranasal (IN) administrations may have fewer side effects thanintraperitoneal (IP) administrations due to a shift in pharmaceuticalresearch to nasal sprays, drops and gels: the nasal route of drugadministration continues to receive increasing attention frompharmaceutical scientists and clinicians because this route circumventshepatic first pass elimination associated with oral delivery, is easilyaccessible and suitable for self-medication. Intranasal administrationalso particularly suits drugs targeting the brain because certain drugsolutions can bypass the blood-brain barrier (BBB) and reach the centralnervous system (CNS) directly from the nasal cavity—uptake of thesedrugs depends on their molecular weight and lipophilicity. Theintranasal delivery increases brain levels of the drug while decreasingsystemic concentrations and thus should have less harmful side effects.

In some embodiments, the present invention provides a method ofprophylactically administering a composition comprising ghrelin toindividuals who are involved in activities, such as contact sports orserving in the armed forces, where there is a possibility of theindividuals suffering mBI. In some embodiments, the present inventionprovides a method for acutely treating individuals who have sufferedmBI. For acute treatments, nasal administration of the compositioncomprising ghrelin may reduce the time for uptake and increase theconcentration of ghrelin that reaches the blood or brain.

For oral administration, such excipients include, e.g., pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesiumcarbonate, and the like. In powders, the carrier is a finely dividedsolid, which is a mixture with the finely divided active component. Intablets, the active component is mixed with the carrier having thenecessary binding capacity in suitable proportions and compacted in theshape and size desired. The powders and tablets contain from one toabout seventy percent of the active compound. Suitable carriers aremagnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.The term “preparation” is intended to include a composition comprisingan active compound disclosed herein with encapsulating material ascarrier providing a capsule in which the active component, with orwithout carriers, is surrounded by a carrier, which is in associationwith it. Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be as solid forms suitablefor oral administration.

Also included are solid form preparations, which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

Other forms suitable for oral administration include liquid formpreparations including emulsions, syrups, elixirs, aqueous solutions,aqueous suspensions, toothpaste, gel dentifrice, chewing gum, or solidform preparations which are intended to be converted shortly before useto liquid form preparations. Emulsions may be prepared in solutions inaqueous propylene glycol solutions or may contain emulsifying agentssuch as lecithin, sorbitan monooleate, or acacia. Aqueous solutions canbe prepared by dissolving the active component in water and addingsuitable colorants, flavors, stabilizing and thickening agents. Aqueoussuspensions can be prepared by dispersing the finely divided activecomponent in water with viscous material, such as natural or syntheticgums, resins, methylcellulose, sodium carboxymethylcellulose, and otherwell-known suspending agents. Solid form preparations include solutions,suspensions, and emulsions, and may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

The administration of ghrelin is based on suitable dosing regimens thattake into account factors well-known in the art including, e.g., type ofsubject being dosed; age, weight, sex and medical condition of thesubject; the route of administration; the renal and hepatic function ofthe subject; the desired effect; and the particular compound employed.Optimal precision in achieving concentrations of drug within the rangethat yields efficacy without toxicity requires a regimen based on thekinetics of the drug's availability to target sites. This involves aconsideration of the distribution, equilibrium, and elimination of adrug.

In some embodiments, ghrelin composition is administered subcutaneously.In some embodiments, ghrelin composition is administered as a bolus,wherein the administration form may be any suitable parenteral form.

Pharmaceutical compositions for parenteral administration includesterile aqueous and non-aqueous injectable solutions, dispersions,suspensions or emulsions, as well as sterile powders to be reconstitutedin sterile injectable solutions or dispersions prior to use. Othersuitable administration forms include suppositories, sprays, ointments,creams, gels, inhalants, dermal patches, implants, pills, tablets,lozenges and capsules.

A typical non-limiting dosage is in a concentration equivalent to from10 ng to 10 mg ghrelin per kg bodyweight. In some embodiments, ghrelinis administered in a concentration equivalent to from about 0.1 μg toabout 1 mg ghrelin per kg bodyweight, such as from about 0.5 μg to about0.5 mg ghrelin per kg bodyweight, such as from about 1.0 μg to about 0.1mg ghrelin per kg bodyweight, such as from about 1.0 μg to about 50 μgghrelin per kg bodyweight, such as from about 1.0 μg to about 10 μgghrelin per kg bodyweight.

In some embodiments, an intravenous injection of ghrelin is employed.The administration route must ensure that the non-degraded, bioactiveform of the peptide will be the dominating form in the circulation,which will reach and stimulate the ghrelin receptors in order to obtainthe maximum effect of ghrelin treatment on mBI. In some embodiments,ghrelin is administered within about 30 minutes of the incident thatresults in mBI. In some embodiments, the ghrelin is administered withinabout 30 minutes to about 2 hours of the incident that results in mBI.In some embodiments, ghrelin is administered within about 30 minutes toabout 6 hours of the incident that results in mBI. In some embodiments,ghrelin is administered within about 30 minutes to about 12 hours of theincident that results in mBI.

Ghrelin compositions may be formulated for parenteral administration(e.g., by injection, for example bolus injection or continuous infusion)and may be presented in unit dose form in ampules, pre-filled syringes,small volume infusion or in multi-dose containers with an addedpreservative. The compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, for examplesolutions in aqueous polyethylene glycol. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution for constitution beforeuse with a suitable vehicle, e.g., sterile, pyrogen-free water. Aqueoussolutions should be suitably buffered if necessary, and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theaqueous solutions are particularly suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. Thesterile aqueous media employed are all readily available by standardtechniques known to those skilled in the art.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions comprising the activeingredient that are adapted for administration by encapsulation inliposomes. In all cases, the ultimate dosage form must be sterile, fluidand stable under the conditions of manufacture and storage. Sterileinjectable solutions are prepared by incorporating ghrelin orpharmaceutical acceptable salt thereof in the required amount in theappropriate solvent with various of the other ingredients enumeratedabove, as required, followed by, e.g., filter sterilization.

Ghrelin compounds can also be delivered topically. Regions for topicaladministration include the skin surface and also mucous membrane tissuesof the rectum, nose, mouth, and throat. Compositions for topicaladministration via the skin and mucous membranes should not give rise tosigns of irritation, such as swelling or redness.

Ghrelin compounds may include a pharmaceutical acceptable carrieradapted for topical administration. Thus, the composition may take theform of, for example, a suspension, solution, ointment, lotion, cream,foam, aerosol, spray, suppository, implant, inhalant, tablet, capsule,dry powder, syrup, balm or lozenge. Methods for preparing suchcompositions are well known in the pharmaceutical industry.

Ghrelin compounds may be administered transdermally, which involves thedelivery of a pharmaceutical agent for percutaneous passage of the druginto the systemic circulation of the patient. The skin sites includeanatomic regions for transdermally administering the drug and includethe forearm, abdomen, chest, back, buttock, and the like. Transdermaldelivery is accomplished by exposing a source of the active compound toa patient's skin for an extended period of time. Transdermal patches canadd advantage of providing controlled delivery of a compound complex tothe body. Such dosage forms can be made by dissolving, dispersing, orotherwise incorporating ghrelin compound in a proper medium, such as anelastomeric matrix material. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate of such fluxcan be controlled by either providing a rate-controlling membrane ordispersing the compound in a polymer matrix or gel.

Kits

Ghrelin compositions may be administered alone or in combination withpharmaceutically acceptable carriers or excipients, in either single ormultiple doses. The formulations may conveniently be presented in unitdosage form by methods known to those skilled in the art. The compoundscan be provided in a kit. Such a kit typically contains an activecompound in dosage forms for administration. The kit comprises an amountof dosage units corresponding to the relevant dosage regimen. In someembodiment, the kit comprises a pharmaceutical composition comprising aghrelin compound or a pharmaceutically acceptable salt thereof andpharmaceutically acceptable carrier, vehicles and/or excipients, saidkit having multiple dosage units. The dosage units comprise an amount ofghrelin or a salt thereof equivalent to from about 0.3 μg to about 600mg ghrelin, such as from about 2.0 μg to about 200 mg ghrelin, such asfrom about 5.0 μg to about 100 mg ghrelin, such as from about 10 μg toabout 50 mg ghrelin, such as from about 10 μg to about 5 mg ghrelin,such as from about 10 μg to about 1.0 mg ghrelin.

The kit contains instructions indicating the use of the dosage form toachieve a desirable affect and the amount of dosage form to be takenover a specified time period. Accordingly, in one embodiment the kitcomprises instructions for administering the pharmaceutical composition.In particular said instructions may include instructions referring toadministration of said pharmaceutical composition after mBI orconcussion, or at the most about 12 hours after the incident causing mBIor concussion, such as at the most about 6 hours after the incidentcausing mBI or concussion, such as at the most about 3 hours after theincident causing mBI or concussion, such as at the most about 1 hoursafter the incident causing mBI or concussion, such as at the most about30 minutes after the incident causing mBI or concussion, such as at themost about 10 minutes after the incident causing mBI or concussion, suchas at the most about 5 minutes after the incident causing mBI orconcussion.

EXAMPLES

The following Examples are intended to further illustrate certainembodiments of the disclosure and are not intended to limit its scope.

Example 1

Ghrelin administration reduces oxidative burst in inflammatory cellsfollowing mBI. Since no well-accepted animal model exists forconcussions, a very small cerebral lesion that closely mimics mBI isused as a model of mBI. C57/B6 mice anesthetized with 5% isoflurane inoxygen (1.7 L/min) are given 0.3 mg/kg buprenorphine subcutaneously foranalgesia prior to infliction of the mild brain injury. Anesthesia isassessed by paw pinch reflex. After creating a burr hole through thedura with a dental drill, a lesion using a controlled cortical impactor(CCI) is used to create injury 1 mm lateral and posterior to the bregma(5.0 mm/sec at a depth of 1.0 mm).

Animals are separated into three treatment groups: 1) Sham, 2) mBI, and3) mBI plus ghrelin. A variety of doses can be tested depending upon theparticular ghrelin. Ghrelin can be obtained for example from PhoenixPharmaceuticals, Inc. (Burlingame, Calif.). For example, 1 to 50 μg atone or more time points. As one example, treatment with subcutaneousghrelin: 1 dose of 10 μg following mBI and additional dose 10 μg 1 hourafter. Brain tissue is harvested at 2-8 hours, preferably about 6 hourspost-injury. Reactive oxygen species (ROS) is measured by neutrophiloxidative burst in leukocytes isolated from brain tissue as shown inFIG. 1 , which depicts the principle of assaying oxidative burst withininflammatory cells. Dihydrorhodamine 123 (DHR 123) diffuses across thecell membrane. When it encounters reactive oxygen species (ROS), DHR 123is oxidized and fluoresces green. The fluorescence is then measured andpresented as arbitrary fluorescence unit (AFU), wherein higher intensitymeans greater oxidative burst and, therefore greater concentration oramount of ROS. Brain cells are isolated (for example by collagenaseDispase/DNAse and a Percoll gradient with resuspension of the pelletusing 100 pi of HBSS). Oxidative burst, which is a marker for ROS, canbe measured by flow cytometry (e.g., FL1 channel with 488-nm laser).

Oxidative burst is increased in animals following mBI compared tocontrol. The number of animals was three in each group (n=3 animals ineach group). Ghrelin treatment reduced the oxidative burst compared tountreated animal with mBI (FIG. 2 ). FIG. 2 shows that subcutaneousghrelin reduces oxidative burst in inflammatory cells following mBI. Theimpact on mBI when ghrelin is administered can be quantified, forexample, by arbitrary fluorescence unit (AFU) (FIG. 3 ). FIG. 3 depictsquantitation of oxidative burst of ipsilateral whole brain followingmild BI. Oxidative burst increased in animals following mBI compared tocontrol. Ghrelin treatment reduced the oxidative burst compared tountreated mBI (FIG. 2 and FIG. 3 ).

As quantified in FIG. 3 , oxidative burst was increased in animalsfollowing mBI compared to sham control (7963 AFU±2900 (mild BI) vs. 4624AFU±1858 (sham); n=5 animals in each group). Ghrelin treatment reducedthe oxidative burst compared to mBI (7963 AFU±2900 (mild BI) vs. 3257AFU±1031 (mild BI +ghrelin (2 doses)); p-value of 0.048 using Student'st-test).

Example 2

The binding ability of ghrelin to GHS-R can be determined by a bindingassay. Chinese hamster ovary cell line cells, CHO-K1, are prepared toexpress the human recombinant GHS receptor.

The cells can be prepared by any suitable method. One such method caninclude: The cDNA for human growth hormone secretagogue receptor(hGHS-R1a, or ghrelin receptor) is cloned by Polymerase Chain Reaction(PCR) using human brain RNA as a template (Clontech, Palo Alto, Calif.),gene specific primers flanking the full-length coding sequence ofhGHS-R, (S: 5′-ATGTGGAACGCGACGCCCAGCGAAGAG-3′ (SEQ ID NO: 2) and AS:5′-TCATGTATTAATACTAGATTCTGTCCA-3′) (SEQ ID NO: 3), and Advantage 2 PCRKit (Clontech). The PCR product is cloned into the pCR2.1 vector usingOriginal TA Cloning Kit (Invitrogen, Carlsbad, Calif.). The full lengthhuman GHS-R is subcloned into the mammalian expression vector pcDNA 3.1(Invitrogen). The plasmid is transfected into the Chinese hamster ovarycell line, CHO-K1 (American Type Culture Collection, Rockville, Md.), bycalcium phosphate method (Wigler, M et al., Cell 11, 223, 1977). Singlecell clones stably expressing the hGHS-R are obtained by selectingtransfected cells grown in cloning rings in RPMI 1640 media supplementedwith 10% fetal bovine serum and 1 mM sodium pyruvate containing 0.8mg/ml G418 (Gibco, Grand Island, N.Y.).

GHS-R binding assay is performed by homogenizing the CHO-K1 cellsexpressing the human recombinant GHS receptor in 20 ml of ice-cold 50 mMTris-HCl with a Brinkman Polytron (Westbury, N.Y.). The homogenates arewashed twice by centrifugation (39,000 g/10 min), and the final pelletsare resuspended in 50 mM Tris-HCl, containing 2.5 mM MgCl₂, and 0.1%BSA. For the GHS-R binding assay, aliquots (0.4 ml) are incubated with0.05 nM (¹²⁵I)ghrelin ({tilde over ( )}2000 Ci/mmol, Perkin Elmer LifeSciences, Boston, Mass.), with and without 0.05 ml of unlabeledcompeting test peptides. After a 60 min incubation (4° C.), the bound(¹²⁵I)ghrelin is separated from the free by rapid filtration throughGF/C filters (Brandel, Gaithersburg, Md.), which have been previouslysoaked in 0.5% polyethyleneimine/0.1% BSA. The filters then are washedthree times with 5m1 aliquots of 50 mM Tris-HCl and 0.1% bovine serumalbumin, and the bound radioactivity trapped on the filters is countedby gamma spectrometry (Wallac LKB, Gaithersburg, Md.). Specific bindingis defined as the total (¹²⁵I)ghrelin bound minus that bound in thepresence of 1000 nM ghrelin.

Example 3

The functional activity of ghrelin is examined using GHS-R functionalactivity assays in vitro and in vivo. Ghrelin binding to GSH receptorcan mediate intracellular iCa²⁺ mobilization in vitro. Ghrelin may alsobe tested for ability to stimulate or suppress release of growth hormone(GH) in vivo.

Cells expressing human GSH receptor can be used. For example, CHO-K1cells expressing the human GSH receptor are harvested by incubating in a0.3% EDTA/phosphate buffered saline solution (25° C.), and are washedtwice by centrifugation. The washed cells are resuspended inHank's—buffered saline solution (HBSS) for loading of the fluorescentCa²⁺ indicator Fura-2AM. Cell suspensions of approximately 10⁶cells/m1are incubated with 2 μM Fura-2AM for 30 min at about 25° C. UnloadedFura-2AM is removed by centrifugation twice in HBBS, and the finalsuspensions are transferred to a spectrofluorometer (Hitachi F-2000)equipped with a magnetic stirring mechanism and a temperature-regulatedcuvette holder. After equilibration to 37° C., ghrelin is added formeasurement of intracellular Ca²⁺ mobilization. The excitation andemission wavelengths can be, for example, 340 and 510 nm, respectively.

Ghrelin may be tested for its ability to stimulate or suppress releaseof growth hormone (GH) in vivo (Deghenghi, R., et al., Life Sciences,1994, 54, 1321-1328; International Application No. WO 02/08250; each ofwhich is incorporated herein by reference in its entirety). Thus, forexample, in order to ascertain ghrelin's ability to stimulate GH releasein vivo the compound may be injected subcutaneously in 10-day old ratsat a dose of, e.g., 300 mg/kg. The circulating GH may be determined at,e.g., 15 minutes after injection and compared to GH levels in ratsinjected with a solvent control.

It is to be understood that while the present disclosure has beendescribed in conjunction with the above embodiments, that the foregoingdescription and examples are intended to illustrate and not limit thescope of the present disclosure. Other aspects, advantages andmodifications within the scope of the present disclosure will beapparent to those skilled in the art to which the present disclosurepertains.

The present disclosure is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the present disclosure, and any compositions ormethods, which are functionally equivalent are within the scope of thisdisclosure. It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present disclosure without departing from the spirit or scope ofthe disclosure. Thus, it is intended that the present disclosure coverthe modifications and variations of this disclosure provided they comewithin the scope of the appended claims and their equivalents.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. (canceled)
 2. A method of treating mild traumatic brain injury (mTBI)in a subject, comprising administering to the subject a therapeuticallyeffective amount of ghrelin, thereby treating the mTBI.
 3. The method ofclaim 2, wherein the ghrelin has the sequence ofGly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg(SEQ ID NO. 1).
 4. The method of claim 2, wherein the ghrelin ismodified with one or more fatty acids.
 5. The method of claim 4, whereinthe fatty acid is an octanoic acid.
 6. The method of claim 3, whereinthe ghrelin is modified at serine at amino acid position 2 and/or serineat amino acid position 3 of SEQ ID NO.
 1. 7. The method of claim 6,wherein the ghrelin is modified at serine at amino acid position 2and/or serine amino acid position 3 with an octanoic acid.
 8. The methodof claim 7, wherein modification of ghrelin is acylation of ghrelin atserine at amino acid position 2 and/or serine at amino acid position 3with an octanoic acid.
 9. The method of claim 2, wherein the subject isa mammal.
 10. The method of claim 2, wherein ghrelin is administeredwithin not more than about 24 hours of the mTBI.
 11. The method of claim2, wherein the subject has a Glasgow Coma Scale score between 13 and 15prior to administration.
 12. The method of claim 2, wherein the ghrelinis administered in an amount that results in ghrelin blood levels beingat least two times greater than the endogenous ghrelin blood levels ofthe subject.
 13. The method of claim 2, wherein ghrelin is administeredin a single dose.
 14. The method of claim 2, wherein ghrelin isadministered at a dosage from 10 ng/kg per day to 10 mg/kg per day. 15.The method of claim 14, wherein ghrelin is administered at a dosage of 2pg/kg per day.
 16. The method of claim 2, wherein the mTBI comprises aconcussion.
 17. A method for treating metabolic derangements associatedwith mild traumatic brain injury (mTBI) in a subject, comprisingadministering to the subject a therapeutically effective amount ofghrelin, thereby treating the metabolic derangements.
 18. A method fortreating increase in reactive oxygen species associated with mildtraumatic brain injury (mTBI), comprising administering to the subject atherapeutically effective amount of ghrelin, thereby treating theincrease in reactive oxygen species.
 19. A method for improvingneuroconnectivity in the brain of a subject after mild traumatic braininjury (mTBI), comprising administering to the subject a therapeuticallyeffective amount of ghrelin, thereby improving neuroconnectivity in thebrain of the subject.
 20. A method for preventing or reducing theincidence or severity of one or more symptoms of mild traumatic braininjury (mTBI) in a subject at risk of mTBI, comprising administering tothe subject a therapeutically effective amount of ghrelin, therebypreventing or reducing the incidence or severity of the one or moresymptoms.